CN115074246A - Microfluidic organ chip with soluble temporary barrier and preparation method thereof - Google Patents

Microfluidic organ chip with soluble temporary barrier and preparation method thereof Download PDF

Info

Publication number
CN115074246A
CN115074246A CN202210833358.7A CN202210833358A CN115074246A CN 115074246 A CN115074246 A CN 115074246A CN 202210833358 A CN202210833358 A CN 202210833358A CN 115074246 A CN115074246 A CN 115074246A
Authority
CN
China
Prior art keywords
barrier
channel
soluble temporary
microfluidic
screen printing
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.)
Pending
Application number
CN202210833358.7A
Other languages
Chinese (zh)
Inventor
王晓林
李钦宇
王丁
轩连
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Jiaotong University
Original Assignee
Shanghai Jiaotong University
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Shanghai Jiaotong University filed Critical Shanghai Jiaotong University
Priority to CN202210833358.7A priority Critical patent/CN115074246A/en
Publication of CN115074246A publication Critical patent/CN115074246A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • 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
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0887—Laminated structure

Abstract

The invention provides a microfluidic organ chip with a soluble temporary barrier and a preparation method thereof, wherein the organ chip comprises: a glass sheet substrate; the PDMS microfluidic chip layer comprises a central tissue chamber, a first culture solution micro-channel and a second culture solution micro-channel, wherein the first culture solution micro-channel and the second culture solution micro-channel are positioned on two sides of the central tissue chamber; the soluble temporary barrier is formed by water-soluble PVA, and comprises a first barrier arranged between the first culture solution micro-channel and the central tissue chamber and a second barrier arranged between the second culture solution micro-channel and the central tissue chamber; the soluble temporary barrier serves as a guide barrier for hydrogel perfusion, and unobstructed culture solution perfusion is performed on the corresponding microfluidic channel after PVA is dissolved. The invention can realize stable and leakage-free ECM perfusion, is more favorable for realizing the construction of the human body micro-physiological environment on a chip, can provide more sufficient and stable fluid stimulation, and can be compatible with different organ chip designs.

Description

Microfluidic organ chip with soluble temporary barrier and preparation method thereof
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to a microfluidic organ chip with a soluble temporary barrier and a preparation method thereof.
Background
The microfluidic technology is a new technology for researching fluid mechanics at the microscopic scale based on miniaturized devices, and has the advantages of less sample consumption, low cost, portability and the like. Through the cross fusion of different disciplines such as biology, medicine, chemistry, materials and the like, the technology attracts the attention of students in the fields of biology, disease diagnosis, chemical synthesis and the like.
In recent years, a series of new cell culture platforms are created due to the seamless fusion of a continuously innovative microfluidic technology and cell biology and tissue engineering technologies. These platforms can be designed into microfluidic devices, allowing control of fluid parameters, thus creating a dynamic, biomimetic microenvironment. The concept of Organ chips (oocs) has been developed using microfluidic systems to simulate the physiological functions or structures of one or more tissues and organs to provide an in vitro model that more closely approximates the physiology of human organs. The establishment of biological microenvironment and real-time biological monitoring continuously improve the understanding of people on cell behaviors in a biological simulation tissue model, and an organ chip becomes an important means for simulating a complex in-vivo microenvironment and carrying out biochemical analysis, has a great promoting effect on the understanding of tissue and organ physiology, and provides a portable, economic and efficient biomedical tool for disease modeling, drug research and personalized medicine.
For organ chips, the generation of perfusable 3D tissues in vitro is one of the important targets for the reconstruction of reliable models of human organs. In a microscale environment, a biogel material-based 3D extracellular matrix (ECM) has become an important approach for constructing biomimetic 3D tissue models in vitro. Common hydrogels can provide a good culture environment for endothelial cells, multicellular tissues, cell-cell and cell-matrix interactions. The hydrogel serving as a scaffold can simulate a hydrated porous microstructure similar to ECM (extracellular matrix), has the characteristics of high water content, high tissue elasticity, good biocompatibility and the like, and is widely applied to the construction of organoid models. For most organ chips, the advantage of integrating nutrient supply and waste discharge itself provides the necessary conditions for long-term tissue co-culture and microscopic observation, so perfusion 3D tissue culture models are currently commonly used. In addition, within a microfluidic chip, in order to ideally construct a microenvironment similar to the size, biochemical factors, and spatial distribution of tissues in vivo, it is important to achieve stable hydrogel patterning using fluid mechanics.
Currently, the most common method of conventional ECM patterning is to design multifunctional physical barriers, such as micropillar arrays or phase-guided (phase-guide) microstructures. These barriers act as capillary burst valves, allowing stable gel loading under gel loading driving forces less than the threshold pressure of gel leakage. In the previous report, based on the design principle of micropillar arrays, M.B.Chen, J.A.Whisler, J.S.Jeon et al in Integrated Biology,2013,5(10): 1262-. Human Umbilical Vein Endothelial Cells (HUVECs) form a microvascular network in the gel matrix, which interacts with lung fibroblasts (NHLFs) on the central culture fluid microchannel through the area between the micropillar arrays. This model demonstrates the effect of inflammatory cytokine stimulation on endothelial barrier function, as well as a positive correlation between metastatic potential of different tumor cell lines and their extravasation capacity. In phase-guided theory, the liquid meniscus in a channel or chamber is subject to capillary forces, which depend on the geometry and material properties of the microstructure. Capillary forces can be used to control the advancement of the liquid meniscus by creating chambers with varying surface characteristics or geometries, based on a phase-guided design, s.j.
Figure BDA0003746429820000021
Joore et al Lab on aChip,2013,13(18): 3548-. Since the phase guide is only a barrier structure below one quarter of the channel height, free communication between channels can be maintained by diffusion, providing nutrients to tissue cells, oxygen, and scavenging waste metabolites. Although both of the above-mentioned designs are easy to prepare, the presence of an internal barrier will inevitably reduce the contact area between the culture broth and the ECM and is likely to trap air bubbles, thereby affecting cell behavior. In addition, the presence of these obstacles can disrupt the integrity of the monolayer formed at the ECM/broth interface, leading to a loss of basement membrane quality, which is detrimental to the co-culture of the vascular layer with other tissue layers.
In order to overcome the adverse effects of barriers such as micropillar arrays and phase guidance on the micro-physiological environment, it is important to develop temporary or virtual barriers to achieve a reliable closed ECM structure. Creation of a free standing polymer film by an interfacial polymerization process was proposed by the following description of "Visualization and characterization of interfacial polymerization layer formation" in Lab on a Chip,2015,15(2): 575-. The polymerization reaction produces a nanoporous temporary polyamine film and forms two microchannels. However, removing this layer requires a chemical reaction, which inevitably affects the cell state and behavior. J. Pei, Q.Sun, Z.Yi et al, Journal of micromechanics and Microengineering,2020,30(3):035005, written "Recoverable elastic barrier for robust hydraulic patterning with inorganic flow profiles for organic-on-a-chip applications", designed a novel Recoverable elastic barrier model that enables hydrogel patterning by a strategy that generates a controllable virtual barrier, and that does not allow the gel to leak out of the central tissue chamber to the side microchannels, enabling more uniform stimulation of cultured cells/tissues. However, the manufacturing process of the microfluidic device is time consuming and the actuation of the elastic barrier requires the cooperation of external fluid control devices.
In summary, there are inevitable problems in terms of tissue culture efficiency, operation difficulty, barrier construction, etc. for methods of generating capillary force and temporary/virtual barrier guiding gel by using microstructures (micro-pillars, phase guiding, etc.), and innovative solutions are needed to improve gel patterning to realize cell/tissue culture of microfluidic platforms.
Disclosure of Invention
In view of the drawbacks of the prior art, it is an object of the present invention to provide a microfluidic organ chip with a soluble temporary barrier and a method for preparing the same.
According to an aspect of the present invention, there is provided a microfluidic organ chip having a soluble temporary barrier, comprising:
a glass sheet substrate for providing support;
the PDMS microfluidic chip layer is positioned on the glass sheet substrate and comprises a central tissue cavity, a first culture solution micro-channel and a second culture solution micro-channel, wherein the first culture solution micro-channel and the second culture solution micro-channel are positioned on two sides of the central tissue cavity, and the central tissue cavity is used for filling hydrogel;
a soluble temporary barrier on the glass substrate, the soluble temporary barrier formed from water-soluble PVA, the soluble temporary barrier comprising a first barrier disposed between the first culture fluid microchannel and the central tissue chamber and a second barrier disposed between the second culture fluid microchannel and the central tissue chamber; the soluble temporary barrier serves as a guide barrier for hydrogel perfusion, and barrier-free culture solution perfusion is carried out on the corresponding microfluidic channel after PVA is dissolved.
According to another aspect of the present invention, there is provided a method for preparing the above microfluidic organ chip having a soluble temporary barrier, the method comprising:
providing a glass sheet substrate, and tightly attaching a screen printing mask on the glass sheet substrate;
completely filling the meshes of the screen printing mask with a PVA solution;
after the PVA solution is solidified, separating the screen printing mask from the glass sheet substrate to form a soluble temporary barrier firmly fixed on the surface of the glass sheet substrate;
and providing a PDMS microfluidic chip layer, aligning one surface with the pattern of the PDMS microfluidic chip layer with the soluble temporary barrier, and bonding the surface with the glass sheet substrate to obtain the microfluidic organ chip with the soluble temporary barrier.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a patterning manufacturing method which uses nontoxic and water-soluble PVA powder as a manufacturing material of a temporary barrier, experiments show that the powder can form viscous liquid when dissolved in water, and can be completely dissolved in water again after being solidified, and a screen printing technology is used as the temporary barrier; in the gel perfusion experiment of the organ chip, the soluble temporary barrier structure can effectively realize the path guidance of the gel and completely dissolve after the culture medium is perfused. The organ chip of the invention not only completes the patterning of the hydrogel, but also achieves the purpose of barrier-free culture solution perfusion after dissolution.
3. The present invention is capable of giving a more adequate and uniform fluid stimulation to the extracellular matrix than traditional physical barrier designs. In the vascularization experiments, the growth of blood vessels in the chip proves that the organ chip has a micro-physiological environment more suitable for cell/tissue culture. In the angiogenesis experiment, the sprouting branches invade the ECM anywhere along the soluble temporary barrier to dissolve, and are more suitable for angiogenesis than the gap between two adjacent microcolumns in the microcolumn array. The organ chip is more beneficial to realizing the construction of the human body micro-physiological environment, can provide sufficient fluid stimulation and is beneficial to realizing barrier-free contact in the process of multi-tissue co-culture, and the soluble temporary barrier design of the invention can be compatible with different organ chips.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIG. 1 is a schematic flow chart of a method for preparing a microfluidic organ chip with a soluble temporary barrier according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a PDMS microfluidic chip layer according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of DTB solubility and hydrogel patterning tests in one embodiment of the invention;
FIG. 4 is a schematic illustration of blood vessel growth in a single chamber DTB microfluidic organ chip according to an embodiment of the present invention;
FIG. 5 is a schematic diagram of the blood vessel growth of a DTB micro-fluidic organ chip with two equal-size chambers according to an embodiment of the present invention;
FIG. 6 is a schematic diagram showing the growth of blood vessels on day 6 of a non-uniform size dual-chamber DTB microfluidic organ chip and a uniform size anisotropic dual-chamber DTB microfluidic organ chip according to an embodiment of the present invention;
FIG. 7 is a graphical representation of the ability of DTBs to restrict hydrogel patterning in nonlinear configurations in accordance with an embodiment of the invention;
FIG. 8 is a schematic illustration of confocal imaging and particle perfusion performed on the generated vascular network in accordance with an embodiment of the present invention;
wherein, in the figure: 1 is PVA solution, 2 is screen printing mask, 3 is glass sheet substrate, 4 is scraper, 5 is soluble temporary barrier, 6 is PDMS micro-fluidic chip layer, 7 is hydrogel, 8 is culture solution micro-channel, R 1 Is a first culture solution micro-channel, R 2 Is a central tissue chamber, R 3 Is a second culture solution micro-channel, V 1 Loading port for first broth, V 2 Loading port for second broth, V 3 Load port for third broth, V 4 Load port for fourth broth, V 5 Loading a first hydrogel with a port, V 6 A first hydrogel loading port.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention. In the description of the embodiments of the present invention, it should be noted that the terms "first", "second", and the like in the description and the claims of the present invention and the drawings described above are used for distinguishing similar objects and not necessarily for describing a particular order or sequence. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in sequences other than those illustrated or described herein.
In the description of the embodiment of the present invention, the DTB, DTB structure, soluble temporary barrier, and soluble temporary barrier all have the same meaning, the ECM represents an extracellular matrix, the PDMS represents polydimethylsiloxane, the PVA represents polyvinyl alcohol, the DTB organ chip, the DTB microfluidic organ chip, the organ chip with soluble temporary barrier, and the microfluidic organ chip with soluble temporary barrier have the same meaning, and the microfluidic channel and the culture solution microchannel have the same meaning.
An embodiment of the present invention provides a microfluidic organ chip with a soluble temporary barrier, which is used for patterning an Extracellular Matrix (ECM) of the organ chip, and referring to fig. 1, the organ chip includes: a glass sheet substrate 3 for providing support and a layer of PDMS (polydimethylsiloxane) microfluidic chip and a soluble Temporary Barrier 5 (DTB) on the glass sheet substrate 3; wherein the PDMS microfluidic chip layer 6 comprises a central tissue chamber R 2 And in the central tissue chamber R 2 The first culture solution micro flow channel R on both sides of 1 And a second culture solution micro flow channel R 3 Central tissue chamber R 2 For perfusion of the hydrogel; the soluble temporary barrier 5 is formed of water-soluble PVA (polyvinyl alcohol), and the soluble temporary barrier 5 is provided in the first culture solution micro flow channel R 1 And a central tissue chamber R 2 The first barrier and the micro-channel R arranged in the second culture solution 3 And a central tissue chamber R 2 A second barrier therebetween; the soluble temporary barrier 5 is used as a guide barrier for hydrogel perfusion, and is used for carrying out barrier-free culture solution perfusion on the corresponding microfluidic channel after PVA is dissolved,namely, the first culture solution micro-channel R is arranged after the first barrier is dissolved 1 Perfusion of the culture solution without barrier is carried out, and after the second barrier is dissolved, the second culture solution micro-channel R is arranged 3 Culture perfusion was performed without obstruction.
The structure of the soluble temporary barrier 5 is matched with that of the PDMS microfluidic chip layer 6, in some specific embodiments, the first barrier and the second barrier are both in a strip shape, and the first barrier is positioned in the first culture solution micro-channel R 1 Inside, the second barrier is arranged in the second culture solution micro-channel R 3 Inner, the distance between the first barrier and the second barrier is larger than the central tissue chamber R 2 Width W of 2 The size is 100-150 mu m, so as to limit ECM to form an ideal patterning effect in the range of the central tissue chamber R2, and the bonding of the PDMS microfluidic chip layer 6 and the glass sheet substrate 3 is not affected, thereby not only ensuring the success rate of the PDMS microfluidic chip layer 6 and the glass sheet substrate 3, but also improving the success rate of hydrogel perfusion.
In some embodiments, the first barrier has a length greater than the central tissue chamber R 2 And a first culture solution micro-channel R 1 The width of the opening at the intersection, the length of the second barrier being greater than the central tissue chamber R 2 And a second culture solution micro-channel R 3 The width of the opening at the intersection; preferably, the central tissue chamber R 2 And a first culture solution micro-channel R 1 Width of opening at intersection with central tissue chamber R 2 And a second culture solution micro-channel R 3 The width of the opening at the intersection is the same, as W in FIG. 2 1 Shown; the first barrier and the second barrier both have a length greater than W 1 To prevent the hydrogel from leaking into the microfluidic channels on both sides.
In some embodiments, the height of the first barrier is smaller than the first culture liquid micro flow channel R 1 The height of the second barrier is smaller than that of the second culture solution micro flow channel R 3 To limit ECM to the central tissue chamber R 2 The ideal patterning effect is formed within the range, and the bonding of the PDMS microfluidic chip layer 6 and the glass sheet substrate 3 is not affected. Preferably, the first culture solution micro flow channel R 1 A second culture solution micro-channel R 3 And a central tissue chamber R 2 The height of (a) is 180-200 μm; the heights of the first barrier and the second barrier are 100-120 μm.
In some embodiments, the first barrier has a width smaller than that of the first culture liquid micro flow channel R 1 The width of the second barrier is smaller than that of the second culture solution micro-channel R 3 Preferably, the width of the first barrier and the second barrier is designed to be 100-200 μm, the first culture liquid micro flow channel R 1 And a second culture solution micro-channel R 3 The width of (D) is designed to be 350-500 μm.
In other embodiments, the shape and size of the PDMS microfluidic chip layer 6 can be customized; the shape and size of the soluble temporary barrier 5 can be individually designed according to the PDMS microfluidic chip layer 6, and can be divided into single or multiple tissue chambers.
In the microfluidic organ chip with the soluble temporary barrier according to the above embodiments of the present invention, the soluble temporary barrier can achieve stable and leak-free ECM perfusion, and can achieve both single-chamber or multi-chamber and regular or irregular ECM patterning, as shown in fig. 3 and 7, the shape of the soluble temporary barrier is a straight line or a curve, and can be set according to actual needs. In addition, the DTB micro-fluidic organ chip is more beneficial to realizing the construction of the human body micro-physiological environment on chip, can provide more sufficient and stable fluid stimulation and can be compatible with different organ chip designs. By constructing three-dimensional vascularized microtissue based on different vascularization mechanisms on a DTB microfluidic organ chip, the robustness and flexibility of the soluble temporary barrier for ECM patterning is verified, as well as the effectiveness of closer to mimicking human interstitial flow stimulation.
The embodiment of the present invention further provides a method for preparing the microfluidic organ chip with the soluble temporary barrier, and with reference to fig. 1, the method includes:
s1, providing a glass sheet substrate 3, and tightly attaching the screen printing mask 2 on the glass sheet substrate 3;
s2, completely filling the meshes of the screen printing mask 2 with the PVA solution 1;
s3, after the PVA solution 1 is solidified, separating the screen printing mask 2 from the glass sheet substrate 3 to form a soluble temporary barrier 5 firmly fixed on the surface of the glass sheet substrate 3;
and S4, providing a PDMS microfluidic chip layer 6, aligning one patterned surface of the PDMS microfluidic chip layer 6 with the soluble temporary barrier 5 under a microscope, and bonding the aligned surface with the glass sheet substrate 3 to obtain the microfluidic organ chip with the soluble temporary barrier.
In some specific embodiments, step S1 includes: the stainless steel screen printing template is micro-machined into a hollow structure matched with the shape of the soluble temporary barrier 5, such as a strip-shaped hollow structure, through laser cutting, the screen printing mask 2 is formed, the magnet is placed below the glass sheet substrate 3, the screen printing mask 2 is tightly attached to the glass sheet substrate 3, and PVA leakage during screen printing is prevented.
The PVA solution 1 may be classified into low, medium and high viscosity according to viscosity, and the PVA solution 1 having higher viscosity is suitable for a screen printing process. In some embodiments, step S2 includes:
s21, slowly pouring PVA powder into distilled water, wherein the mass ratio of the PVA powder to the distilled water is 1:5-1:8, uniformly stirring, sealing by using a preservative film, and forming a PVA solution 1 with the viscosity of 44-56mPa & S after bubbles in the solution are dissolved;
s22, the PVA solution 1 is placed on the screen printing mask 2, and the squeegee 4 is moved on the screen printing mask 2 to fill the openings of the screen printing mask 2 with the PVA solution 1, and this operation is repeated until the openings on the screen printing mask 2 are completely filled.
In some specific embodiments, step S3 includes:
s31, under the condition that the glass sheet substrate 3 is kept to be tightly attached to the screen printing mask 2, putting the glass sheet substrate 3 and the screen printing mask into an oven with the temperature of 55-75 ℃ for 5-10min to solidify the PVA solution 1;
s32, the magnet is removed and the screen printing stencil is gently separated from the glass sheet substrate 3, leaving the soluble temporary barrier 5 on the surface of the glass sheet substrate 3.
The preparation method in the embodiment develops the manufacturing process of the soluble temporary barrier based on the PVA material for screen printing, and proposes that the screen printing technology is used asThe method is a patterning manufacturing method of the temporary barrier, uses nontoxic and water-soluble PVA powder as a manufacturing material of the temporary barrier, and is simple and convenient. Unlike conventional designs, there is no physical barrier like a micropillar array to separate the tissue chamber from the microfluidic channel, in the central tissue chamber R 2 And the opening with larger size is arranged between the culture solution micro-channel, so that the DTB structure can effectively guide the path of the hydrogel, and after the PVA is completely dissolved in the perfusion culture solution, the contact area between the hydrogel in the tissue cavity and the lateral culture solution can be greatly increased, thereby being beneficial to the nutrient supply in the subsequent cell culture.
Referring to FIG. 2, the PDMS microfluidic chip layer 6 of FIG. 1 comprises a central tissue chamber R 2 And with the central tissue chamber R 2 Communicating first hydrogel loading port V 5 A second hydrogel loading port V 6 (ii) a First culture solution micro-channel R 1 And a micro flow channel R for the first culture solution 1 A first culture solution loading port V communicated with 1 A second culture solution loading port V 2 (ii) a Second culture solution micro-channel R 3 And a second culture solution micro flow channel R 3 A communicated third culture solution loading port V 3 A fourth culture solution loading port V 4 . In some specific embodiments, the step S4 is to provide the PDMS microfluidic chip layer 6, wherein the PDMS microfluidic chip layer 6 is prepared by using a standard photolithography process, and the method for preparing the PDMS microfluidic chip layer 6 includes:
s41, preparing a mask: designing a mask to match the structure size of the soluble temporary barrier and the PDMS pattern;
s42, silicon wafer cleaning: cleaning a single polished silicon wafer in an acetone solution, an ethanol solution and deionized water in sequence until the polished surface of the silicon wafer is clean and free of stains, drying the cleaned silicon wafer by using a nitrogen gun, and baking the silicon wafer on a hot plate until water vapor is removed;
s43, glue homogenizing and pre-baking: spin-coating photoresist on the whole surface of a silicon wafer, placing the silicon wafer on a spin coater for spin-coating, carrying out pre-baking treatment, and then waiting for the temperature to be reduced to room temperature in a step manner;
s44, photoetching and postbaking: exposing the photoresist to ultraviolet light through a mask plate to obtain the photoresist, and then carrying out post-baking treatment;
s45, developing and silanizing: taking out the silicon wafer cooled to room temperature, immersing the silicon wafer in a developing solution in a yellow region, then putting the silicon wafer into an isopropanol solution to remove the developing solution, and then washing the silicon wafer in deionized water; blow-drying by a nitrogen gun, and then performing silanization surface treatment;
s46, inverted-mode PDMS microfluidic chip layer: and mixing the PDMS prepolymer with a curing agent thereof, pouring the mixture onto the surface of the photoresist, carrying out curing treatment, and carrying out demoulding and punching operations to obtain the PDMS microfluidic chip layer 6.
Preferably, the preparation method of the PDMS microfluidic chip layer 6 includes:
s41, preparing a mask: the PDMS pattern structure matched with the size of the soluble temporary barrier structure is designed by using AutoCAD, the structure of the PDMS microfluidic chip layer 6 is shown in FIG. 2, and the shapes and the sizes of the two structures can be customized according to needs. Micro-channel R for culture solution in mask 1 、R 3 Tissue chamber R 2 Hydrogel loading port V 5 、V 6 And a culture solution loading port V 1 、V 2 、V 3 、V 4 The transparent mask is set, and the rest areas are set to be black to prepare the mask.
S42, silicon wafer cleaning: and cleaning the single polished silicon wafer in an acetone solution, an ethanol solution and deionized water for 8min by using an ultrasonic cleaning machine in sequence until the polished surface of the silicon wafer is clean and free of stains. The cleaned silicon wafer was blow dried using a nitrogen gun and baked on a hot plate at 180 ℃ until water vapor was removed.
S43, glue homogenizing and pre-baking: spin-coating SU-8 photoresist on the whole surface of a silicon wafer, placing the silicon wafer on a spin coater for spin coating to obtain 180-thick SU-8 photoresist with the thickness of 200 μm, placing the SU-8 photoresist in an oven at 60 ℃ for 10min, then baking the SU-8 photoresist at 95 ℃ for 4h, and finally waiting for the temperature to be reduced to room temperature in a step manner.
S44, photoetching and postbaking: and exposing the photoresist to ultraviolet irradiation through a mask to carry out photoetching on the SU-8 photoresist, and placing the SU-8 photoresist in an oven at the temperature of 95 ℃ for 30 min.
S45, developing and silanizing: taking out the silicon wafer cooled to room temperature, immersing the silicon wafer in a developing solution in a yellow light area for 5-8min, then putting the silicon wafer into an isopropanol solution to remove the developing solution, and then washing the silicon wafer in deionized water. And blow-dried with a nitrogen gun, followed by silanization surface treatment.
S46, inverted-mode PDMS microfluidic chip layer: mixing the PDMS prepolymer and the curing agent thereof according to the proportion of 10:1, pouring the mixture onto the surface of SU-8 photoresist, putting the mixture into a 70 ℃ oven for 2h to cure the mixture, and finally cutting PDMS to obtain the needed PDMS microfluidic chip.
With continued reference to fig. 1, in some preferred embodiments, a method of preparing a microfluidic organ chip with a soluble temporary barrier comprises:
s1, as shown in fig. 1 (a), a stainless steel plate material having a thickness of 120 μm is micro-machined into a long strip-shaped hollow structure by laser cutting to be a screen printing mask 2, and is closely attached to a glass substrate 3 with a magnet to prevent liquid leakage at the time of screen printing.
S2, as shown in fig. 1 (b), the PVA solution 1 having a high viscosity is placed on the screen printing mask 2, and the doctor blade 4 is moved to fill the open mesh with the PVA solution 1. This process can be repeated several times until the meshes of the screen printing mask 2 are completely filled, and then the whole is placed in an oven at 60 ℃ for 7 min. In this step, PVA powder can be classified into low viscosity, medium viscosity and high viscosity according to the viscosity. And slowly pouring a proper amount of medium-viscosity PVA powder into distilled water, wherein the mass ratio of PVA to distilled water is 1:5, uniformly stirring, sealing by using a preservative film, dissolving bubbles in the solution, and then using for silk-screen printing, wherein the obtained PVA solution 1 has high viscosity and is suitable for a silk-screen printing process.
S3, as shown in fig. 1 (c), after the PVA is cured, the magnet is removed and the screen printing mask 2 is separated from the glass substrate 3, and the soluble temporary barrier 5 can be firmly fixed to the surface of the glass substrate 3.
S4, as shown in fig. 1 (d), performing oxygen plasma surface treatment on the PDMS microfluidic chip layer 6 and the glass substrate 3, aligning one patterned surface of the PDMS microfluidic chip layer 6 with the temporary solvent barrier 5, bonding the patterned surface to the glass substrate 3, and placing the bonded chip on a hot plate at 152 ℃ for 10min to enhance the bonding effect between the two.
As shown in FIG. 1 (e), the gap between the two temporary barriers should be designed slightly larger than the tissue cavity R in FIG. 2 2 To prevent the hydrogel from leaking to the side culture solution micro flow channel R 1 、R 3 In (1). In addition, to facilitate better alignment during bonding, the length of the temporary barrier 5 should be designed to be greater than the tissue chamber R 2 The width of the opening.
In the preparation method of the microfluidic organ chip with the soluble temporary barrier in the embodiment of the invention, the soluble material is patterned on the glass sheet substrate by using a screen printing process, and the glass sheet substrate is bonded with the Polydimethylsiloxane (PDMS) microfluidic chip after the soluble material is solidified, so that the DTB microfluidic organ chip is prepared. Compared with the conventional method of patterning ECM by adopting physical barriers such as a micro-column array and the like in the traditional microfluidic organ chip, the embodiment of the invention can realize barrier-free fluid perfusion and increase the effective contact area between ECM and fluid, which can directly determine the uniformity and strength of cells/tissues stimulated by the fluid in ECM and the integrity of a basement membrane on an ECM interface. The invention can solve the key technical problems of the conventional general hydrogel patterning method, particularly aims at sealing a microfluidic chip, so that the method is expected to become a new paradigm for developing novel in-vitro organ chip models and other microfluidic-based cell/tissue culture analysis.
Referring to fig. 3, in order to verify the feasibility of a soluble temporary barrier design for patterning of an extracellular matrix of an organ chip and a preparation method thereof, a DTB solubility and hydrogel patterning test was performed, comprising the following steps:
s100, as shown in fig. 3 (a) and (b), the chip cross-sectional views after the screen printing step and the bonding step correspond to the schematic diagrams, respectively, and fig. 3 (e) and (f) are enlarged real object views under a microscope. After the glass substrate 3 is bonded with the PDMS microfluidic chip layer 6, a first culture solution micro-channel R 1 A second culture solution micro-channel R 3 Clearly visible, without being blocked by the soluble temporary barrier 5, the subsequent culture perfusion is not affected either. Here, even though the thickness of the soluble temporary barrier 5 is half the height of the tissue cavityThe hydrogel 7 may also smoothly follow the soluble temporary barrier 5 and be firmly confined to the central tissue chamber R 2 Without overflowing to the side surface of the first culture solution micro flow channel R against the meniscus pinning effect 1 And a second culture solution micro flow channel R 3 。
S200, hydrogel 7 mixed with Human Umbilical Vein Endothelial Cells (HUVEC) and human lung fibroblasts (NHLF) was perfused into the central tissue chamber R as shown in FIG. 3 (c) 2 Also, due to the high water content of the hydrogel, the soluble temporary barrier 5 is partially dissolved after 1min of close contact therewith, and the actual effect is shown in fig. 3 (g). Since the soluble temporary barrier 5 is dissolved for a longer period of time than the time required for the hydrogel 7 to polymerize, it does not affect the central tissue cavity R 2 Patterning the hydrogel therein.
S300, as shown in fig. 3 (d), after the hydrogel 7 is solidified, the cell culture solution is poured into the culture solution micro flow channel 8, and the soluble temporary barrier 5 is completely dissolved after 1min, so that the ECM and the culture solution are in barrier-free contact, and the actual effect is shown in fig. 3 (h).
Thus, in addition to stable ECM patterning without leakage, this design also provides a more adequate nutrient supply and fluid stimulation to the cultured cells/tissues. The DTB structure spontaneously dissolves in a liquid environment without the need for specific chemical reactions or the intervention of external equipment.
The actual culture effect of microfluidic organ chips of different structures is explained below to demonstrate the feasibility of the soluble temporary barrier design and its preparation method for patterning of the extracellular matrix of organ chips.
Referring to fig. 4 (a) - (h), there are photographs of the blood vessel growth in the single-chamber DTB microfluidic organ chip from day 1 to day 13, and the structure corresponds to the chip structures of fig. 3 (e) - (h). Wherein (a) to (d) in FIG. 4 are bright field microscope pictures, and (e) to (h) in FIG. 4 are fluorescence microscope pictures, the scale in the pictures is 500 μm. The tissue chamber was filled with a hydrogel mixed with HUVEC and NHLF, both at an initial seeding concentration of 5X 10 6 /mL。
Referring to FIG. 4, the width of the central tissue chamber R2 is 1mm, the width of the culture fluid micro channel is 350 μm, and under the two-side fluid stimulation, it can be seen that HUVEC begin to show fragmentation growth and form connection under the induction of NHLF on the 5 th day; lumen formation began on day 9 and gradually thickened, creating a 3D capillary network. By day 13 of growth, the vessel density and lumen diameter further increased, and the lumen diameter in local areas could approach 100 μm, which provides excellent channels for drug small molecule delivery. Since the openings between the central tissue chamber R2 and the culture fluid microchannel are large, the distribution and size of the lumen openings are not limited at all, which may better reproduce the capillary network anastomosed in vivo.
Referring to (a) and (b) in fig. 5, the images of the blood vessel growth of the equal-size double-chamber DTB microfluidic organ chip on the 0 th day and the 13 th day are shown, and the scale is 500 μm. As can be seen, similar to the single-chamber structure, by day 13 of blood vessel growth, the capillary network becomes more mature, the lumen size increases, and the vascular structure tends to grow in a direction consistent with the direction of the fluid shear force, so-called mechanical migration, which is a key process of vascular remodeling.
Referring to (a) and (b) in fig. 6, there are respectively the blood vessel growth pictures of the non-equal-size double-chamber DTB micro-fluidic organ chip and the equal-size anisotropic double-chamber DTB micro-fluidic organ chip on day 6, where the scale is 500 μm. It can be seen from the figure that, whatever the structure, 3D capillary networks had formed by day 6 in culture. The results of the angiogenesis experiments performed on the dual chamber demonstrate the effectiveness of the microfluidic organ chip with a soluble temporary barrier and the method for preparing the same in the embodiments of the present invention for the multi-chamber structure design.
To further verify the ability of the DTB to limit hydrogel patterning in non-linear configurations, a circular tissue chamber was divided into three adjacent compartments by two DTBs, as shown in figures 7 (a) - (d). In FIG. 7, (a) and (b) are bright field microscope pictures, and in FIG. 7, (c) and (d) are fluorescence microscope pictures with a scale of 500. mu.m. The hydrogel mixed with HUVEC and NHLF was injected into the upper and lower semicircular tissue chambers, respectively, and the DTB structures on both sides ideally achieved the patterning of the hydrogel, as shown in fig. 7 (c). After the DTB was dissolved, a hydrogel mixed with human brain astrocytoma cells (U87MG) was injected into the central oval tissue chamber, and as the culture time was prolonged, a large number of U87MG cells collectively migrated in a radial arrangement toward the bilateral microvascular network, as shown in fig. 7 (b) and (d). The results of this experiment show that tumor cells with highly metastatic properties are able to migrate gradients of growth factors and nutrients. The tissue chamber may be implemented to interact with the ECM in a non-physical barrier manner to enable the construction of a vascularized tumor platform. In addition, one tissue chamber is accurately and spatially separated through the DTB structure, and three chambers can be formed to achieve co-culture of two-side three-dimensional capillaries and tumor cell tissues so as to simulate the growth condition of human tumor tissues.
To further verify whether the formed microvascular network is three-dimensional and demonstrate the perfusability of the microvascular network, confocal imaging and particle perfusion of the generated vascular network were performed. With reference to fig. 8 (a) and (b), confocal microscopy fluorescence imaging demonstrated three-dimensional vascular distribution at different heights within a thickness of 200 μm, and in addition, confocal images of horizontal (XY plane) and vertical (XZ plane and YZ plane) sections showed that microvessels were formed having hollow tubes with a diameter of about 50 μm.
Referring to fig. 8 (c), fluorescent microparticles having a diameter of 5 μm are introduced into the culture fluid micro channel on one side through the culture fluid loading port with high hydrostatic pressure, and rapidly and easily pass through the microvascular network into the microfluidic channel on the other side without undesired leakage.
To assess the non-physiological leaky nature of the resulting 3D microvessels, 70kDa FITC-dextran with green fluorescence was also perfused into the vessel lumen. Referring to fig. 8 (D), the 3D microvascular network, which is gradually filled with dextran over time, flows from the high pressure microchannel on one side to the low pressure microvascular region on the other side, and no dextran is observed outside the vessel lumen after 10min of perfusion. These experimental results all prove that the microvascular network formed in the DTB organ chip has excellent small molecule substance transport performance, and the tube wall has strong barrier performance. Provides a good platform for researching the drug delivery in blood vessels in vitro and targeting cancer therapy.
The above experiment can prove that the above embodiment of the present invention provides a patterning manufacturing method using a screen printing technology as a temporary barrier, and uses non-toxic and water-soluble PVA powder as a manufacturing material of the temporary barrier. Experiments show that the powder can form viscous liquid when dissolved in water, can be completely dissolved in water again after being solidified, and is suitable for a screen printing process. Meanwhile, in a gel perfusion experiment of the DTB organ chip, the DTB structure can effectively realize the path guidance of the gel and is completely dissolved after a culture medium is perfused. The DTB organ chip not only completes the patterning of the hydrogel, but also achieves the purpose of barrier-free culture solution perfusion after dissolution. Thus, a more adequate and uniform fluid stimulation of the extracellular matrix can be imparted than with conventional physical barrier designs. In the vascularization experiments, the growth of blood vessels in the chip proves that the DTB organ chip has a micro-physiological environment more consistent with cell/tissue culture. In the angiogenesis experiment, the sprouting branch invades ECM anywhere along the DTB lysis, and is more suitable for angiogenesis than the gap between two adjacent microcolumns in the microcolumn array. The DTB organ chip is more beneficial to realizing the construction of the human body-imitated micro-physiological environment, can provide sufficient fluid stimulation, is beneficial to realizing barrier-free contact in the process of multi-tissue co-culture, and can be compatible with different organ chips in the DTB design. The microfluidic organ chip of the embodiment provides innovative application value in the biomedical engineering field, particularly for the related research of in vitro vascularized tumor disease model construction and novel anti-tumor drug screening.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention. The above-described preferred features may be used in any combination without conflict with each other.

Claims (10)

1. A microfluidic organ chip with a soluble temporary barrier, comprising:
a glass sheet substrate for providing support;
the PDMS microfluidic chip layer is positioned on the glass sheet substrate and comprises a central tissue chamber, a first culture solution micro-channel and a second culture solution micro-channel which are positioned on two sides of the central tissue chamber, and the central tissue chamber is used for filling hydrogel;
a soluble temporary barrier on the glass substrate, the soluble temporary barrier formed from water-soluble PVA, the soluble temporary barrier comprising a first barrier disposed between the first culture fluid microchannel and the central tissue chamber and a second barrier disposed between the second culture fluid microchannel and the central tissue chamber; the soluble temporary barrier serves as a guide barrier for hydrogel perfusion, and barrier-free culture solution perfusion is carried out on the corresponding microfluidic channel after PVA is dissolved.
2. The microfluidic organ chip with a soluble temporary barrier according to claim 1, wherein the first barrier is located inside the first culture solution micro channel, the second barrier is located inside the second culture solution micro channel, and a distance between the first barrier and the second barrier is slightly larger than a width of the central tissue chamber.
3. The microfluidic organ chip with a soluble temporary barrier according to claim 1, wherein the length of the first barrier is greater than the width of an opening where the central tissue chamber intersects the first culture fluid micro channel; the length of the second barrier is larger than the width of an opening at the intersection of the central tissue chamber and the second culture solution micro-channel, so that the water gel is prevented from leaking into the micro-fluidic channels on two sides.
4. The microfluidic organ chip with a soluble temporary barrier according to claim 1, wherein the height of the first barrier is smaller than the height of the first culture fluid micro channel, the height of the second barrier is smaller than the height of the second culture fluid micro channel, the width of the first barrier is smaller than the width of the first culture fluid micro channel, and the width of the second barrier is smaller than the width of the second culture fluid micro channel.
5. The microfluidic organ chip with soluble temporary barrier according to claim 4, wherein the height of the first culture fluid micro flow channel, the second culture fluid micro flow channel and the central tissue chamber is 180-200 μm; the heights of the first barrier and the second barrier are 100-120 mu m; the widths of the first barrier and the second barrier are designed to be 100-200 mu m, and the widths of the first culture fluid micro-channel and the second culture fluid micro-channel are designed to be 350-500 mu m.
6. A method for preparing a microfluidic organ chip with a soluble temporary barrier according to any one of claims 1 to 5, comprising:
providing a glass sheet substrate, and tightly attaching a screen printing mask on the glass sheet substrate;
completely filling the meshes of the screen printing mask with a PVA solution;
after the PVA solution is solidified, separating the screen printing mask from the glass sheet substrate to form a soluble temporary barrier firmly fixed on the surface of the glass sheet substrate;
and providing a PDMS microfluidic chip layer, aligning one surface with the pattern of the PDMS microfluidic chip layer with the soluble temporary barrier, and bonding the surface with the glass sheet substrate to obtain the microfluidic organ chip with the soluble temporary barrier.
7. The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 6, wherein the step of closely attaching a screen printing mask on the glass substrate comprises: the stainless steel screen printing template is micro-processed into a hollow structure matched with the shape of the soluble temporary barrier through laser cutting to form a screen printing mask, and the screen printing mask is tightly attached to the glass sheet substrate through a magnet.
8. The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 6, wherein the completely filling the mesh of the screen printing mask with PVA solution comprises:
slowly pouring PVA powder into distilled water, wherein the mass ratio of the PVA powder to the distilled water is 1:5-1:8, uniformly stirring, sealing by using a preservative film, and dissolving bubbles in the solution to form a PVA solution with preset viscosity;
a PVA solution was placed on the screen printing mask, and a squeegee was moved on the screen printing mask to fill the openings of the screen printing mask with the PVA solution, and this operation was repeated until the openings on the screen printing mask were completely filled.
9. The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 7, wherein the step of separating the screen printing mask from the glass sheet substrate after the PVA solution is solidified comprises:
under the condition of keeping the glass sheet substrate and the screen printing mask tightly attached, putting the glass sheet substrate and the screen printing mask into an oven with the temperature of 55-75 ℃ for 5-10min to solidify the PVA solution;
and removing the magnet and slightly separating the screen printing template from the glass sheet substrate, and keeping the soluble temporary barrier on the surface of the glass sheet substrate.
10. The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 6, wherein the PDMS microfluidic chip layer is provided, wherein the PDMS microfluidic chip layer is prepared by a standard photolithography process, and the method for preparing the PDMS microfluidic chip layer comprises:
preparing a mask plate: designing a mask to match the structure size of the soluble temporary barrier and the PDMS pattern;
cleaning a silicon wafer: cleaning a single polished silicon wafer in an acetone solution, an ethanol solution and deionized water in sequence until the polished surface of the silicon wafer is clean and free of stains, drying the cleaned silicon wafer by using a nitrogen gun, and baking the silicon wafer on a hot plate until water vapor is removed;
glue homogenizing and pre-drying: spin-coating photoresist on the whole surface of a silicon wafer, placing the silicon wafer on a spin coater for spin-coating, carrying out pre-baking treatment, and then waiting for the temperature to be reduced to room temperature in a step manner;
photoetching and post-baking: exposing the photoresist to ultraviolet light through a mask plate to obtain the photoresist, and then carrying out post-baking treatment;
developing and silanization: taking out the silicon wafer cooled to room temperature, immersing the silicon wafer in a developing solution in a yellow region, then putting the silicon wafer into an isopropanol solution to remove the developing solution, and then washing the silicon wafer in deionized water; blow-drying by a nitrogen gun, and then performing silanization surface treatment;
inverted mold PDMS microfluidic chip layer: and mixing the PDMS prepolymer with a curing agent thereof, pouring the mixture onto the surface of the photoresist, carrying out curing treatment, and carrying out demoulding and punching operations to obtain the PDMS microfluidic chip layer.
CN202210833358.7A 2022-07-14 2022-07-14 Microfluidic organ chip with soluble temporary barrier and preparation method thereof Pending CN115074246A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210833358.7A CN115074246A (en) 2022-07-14 2022-07-14 Microfluidic organ chip with soluble temporary barrier and preparation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210833358.7A CN115074246A (en) 2022-07-14 2022-07-14 Microfluidic organ chip with soluble temporary barrier and preparation method thereof

Publications (1)

Publication Number Publication Date
CN115074246A true CN115074246A (en) 2022-09-20

Family

ID=83259070

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210833358.7A Pending CN115074246A (en) 2022-07-14 2022-07-14 Microfluidic organ chip with soluble temporary barrier and preparation method thereof

Country Status (1)

Country Link
CN (1) CN115074246A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117250345A (en) * 2023-11-20 2023-12-19 重庆医科大学绍兴柯桥医学检验技术研究中心 In-situ detection method of biomolecules in organ chip

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117250345A (en) * 2023-11-20 2023-12-19 重庆医科大学绍兴柯桥医学检验技术研究中心 In-situ detection method of biomolecules in organ chip
CN117250345B (en) * 2023-11-20 2024-02-13 重庆医科大学绍兴柯桥医学检验技术研究中心 In-situ detection method of biomolecules in organ chip

Similar Documents

Publication Publication Date Title
Moshksayan et al. Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture
Zhang et al. Gel integration for microfluidic applications
Walsh et al. A multipurpose microfluidic device designed to mimic microenvironment gradients and develop targeted cancer therapeutics
Tehranirokh et al. Microfluidic devices for cell cultivation and proliferation
US10544036B2 (en) Microfluidic devices and methods for the extrusion of tubular structures
US9617520B2 (en) Device and method of 3-dimensionally generating in vitro blood vessels
CN111235029B (en) Multifunctional microfluidic chip and preparation method and application thereof
CN103146576B (en) Micro-fluidic chip culture device of cell capable of being stretched mechanically and application thereof
Underhill et al. Bioengineering methods for analysis of cells in vitro
CN111218404A (en) Bionic multi-organ chip and preparation method and application thereof
KR20140063521A (en) Microfluidic system for controlling the concentration of molecules for stimulating a target
JP2011500018A (en) Method for forming a cell structure using a temporary linker in a cage
CN115074246A (en) Microfluidic organ chip with soluble temporary barrier and preparation method thereof
JP2019531731A (en) Three-dimensional (3D) hydrogel patterning in a microfluidic blood vessel model
Ge et al. Development of multi-dimensional cell co-culture via a novel microfluidic chip fabricated by DMD-based optical projection lithography
Xu et al. Development of disposable PDMS micro cell culture analog devices with photopolymerizable hydrogel encapsulating living cells
US11326138B2 (en) Cell culture device and methods of use thereof
CN108504571B (en) Device and method for constructing artificial liver lobule functional unit
CN212316139U (en) Bionic multi-organ chip
KR102171936B1 (en) A method for liquid patterning and cell immobilization in microfluidic platform using surface tension
Tong et al. Automated addressable microfluidic device for minimally disruptive manipulation of cells and fluids within living cultures
Wang et al. Pneumatic mold-aided construction of a three-dimensional hydrogel microvascular network in an integrated microfluidics and assay of cancer cell adhesion onto the endothelium
KR20240030938A (en) Microfluidic device with partially enclosed microfluidic channels and method of forming perfusable vascular network
CN103992948B (en) A kind of micro-nano-fluidic control device for cell migration research
US20210054322A1 (en) Devices and methods for three-dimensional growth of cells

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