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

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 technique

Microfluidics is an emerging technology based on miniaturized devices to study fluid mechanics at the microscopic scale. It has the advantages of less sample consumption, low cost, and portability. Through the cross-integration of different disciplines such as biology, medicine, chemistry, and materials, this technology is attracting the attention of scholars from all walks of life in the fields of biology, disease diagnosis, and chemical synthesis.

In recent years, the continuous integration of innovative microfluidic technology with cell biology and tissue engineering technology has resulted in the birth of a series of new cell culture platforms. These platforms can be designed within microfluidic devices, allowing the control of fluid parameters, resulting in a dynamic, biomimetic microenvironment. Using microfluidic systems to simulate the physiological function or structure of one or more tissues and organs to provide an in vitro model closer to the physiology of human organs, the concept of Organ on a Chip (OoC) was born. The establishment of biological microenvironments and real-time biomonitoring continue to improve people's understanding of cell behavior in biomimetic tissue models. Organ chips have become an important means of simulating complex in vivo microenvironments and conducting biochemical analysis, which is extremely useful in promoting the understanding of tissue and organ physiology. A major advance in providing portable and cost-effective biomedical tools for disease modeling, drug research, and personalized medicine.

For organ-on-a-chip, the generation of perfusable 3D tissue in vitro is one of the important goals for the reconstruction of reliable models of human organs. In the micro-scale environment, 3D extracellular matrix (ECM) based on biogel materials has become an important way to construct 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. As scaffolds, hydrogels can mimic ECM-like hydrated porous microstructures with high water content, high tissue elasticity, and good biocompatibility, and have been widely used in the construction of organoid models. For most organ-on-chips, the advantages of their own integrated nutrient supply and waste discharge provide the necessary conditions for long-term tissue co-culture and microscopic observation, so perfusion 3D tissue culture models are currently commonly used. Furthermore, within the microfluidic chip, in order to ideally construct a microenvironment similar to in vivo tissue size, biochemical factors, and spatial distribution, it is crucial to achieve stable hydrogel patterning using hydrodynamics.

J.Joore等人在Lab on a Chip,2013,13(18):3548-3554上撰文“Microfluidic titerplate for stratified 3D cell culture”，提出一种分层的3D细胞培养平台，其中组织腔室和灌注通道是相邻的，由相位引导分隔且没有物理隔离，弯月面的夹持阻挡作用防止凝胶溢出到侧面的通道。由于相位引导仅仅是低于通道高度四分之一的屏障结构，通道之间的自由交流可以通过扩散得以保持，为组织细胞提供营养、氧气并清除废弃的代谢物。尽管上述两类设计易于制备，但内部屏障的存在将不可避免地减少培养液和ECM之间的接触面积，并且很可能捕获气泡，从而影响细胞行为。此外，这些障碍物的存在会破坏在ECM/培养液界面上形成单细胞层的完整性，导致基底膜质量降低，不利于血管层与其他组织层的共培养。Currently, the most commonly used method for traditional ECM patterning is to design multifunctional physical barriers, such as micropillar arrays or phase-guide microstructures. These barriers act as capillary burst valves to stabilize gel loading when the driving force for gel loading is less than the threshold pressure for gel leakage. In a previous report, based on the design principles of micropillar arrays, MBChen, JAWhisler, JSJeon et al. wrote "Mechanisms of tumor cell extravasation in an in vitromicrovascular network platform" in Integrative Biology, 2013, 5(10):1262-1271 , to develop a microfluidic platform that simulates a three-dimensional microvascular network in vitro. During the gel filling process, the micropillars at the end of each gel area are assisted by surface tension to guide the gel filling and prevent leakage. Human umbilical vein endothelial cells (HUVECs) form a microvascular network in a gel matrix, passing through the area between the micropillar arrays, interacting with lung fibroblasts (NHLFs) on a central medium microchannel. This model demonstrates the effect of inflammatory cytokine stimulation on endothelial barrier function and a positive correlation between the metastatic potential of different tumor cell lines and their extravasation capacity. In phase guidance theory, the liquid meniscus in a channel or chamber experiences capillary forces that 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 properties or geometries, based on phase-guided design, SJTrietsch, GD

J.Joore et al., "Microfluidic titerplate for stratified 3D cell culture" in Lab on a Chip, 2013, 13(18):3548-3554, propose a stratified 3D cell culture platform in which tissue chambers and perfusion The channels are adjacent, separated by phase guides and not physically isolated, and the clamping barrier action of the meniscus prevents gel from spilling into the side channels. Since phase guidance is only a barrier structure that is less than a quarter of the channel height, free communication between channels can be maintained by diffusion, supplying tissue cells with nutrients, oxygen, and scavenging waste metabolites. Although the above two types of designs are easy to prepare, the presence of an internal barrier will inevitably reduce the contact area between the culture medium and the ECM, and will likely trap air bubbles, thereby affecting cell behavior. In addition, the presence of these obstacles disrupts the integrity of the monolayer formed at the ECM/culture medium interface, resulting in reduced basement membrane quality and unfavorable co-culture of the vascular layer with other tissue layers.

To overcome the detrimental effects of barriers such as micropillar arrays and phase guidance on the microphysiological environment, it is crucial to develop temporary or virtual barriers to achieve reliable closed ECM structures. Y.Zhang,N.E.Benes,R.G.H.Lammertink, "Visualization and characterization of interfacial polymerization layer formation" in Lab ona Chip, 2015, 15(2): 575-580, proposed a method to create independent polymers by interfacial polymerization film. The polymerization produced a nanoporous temporary polyamine film and formed two microchannels. However, removing this membrane requires a chemical reaction that inevitably affects cell state and behavior. "Recoverable elastic barrier for robust hydrogel patterning with uniform flow profile for organ-on-a-chip applications" by J. Pei, Q. Sun, Z. Yi et al. in Journal of Micromechanics and Microengineering, 2020, 30(3):035005 , a new recoverable elastic barrier model was designed, and the hydrogel patterning was realized by the strategy of generating a controllable virtual barrier, and the gel would not leak from the central tissue chamber to the side microchannels, which could effectively affect the cultured cells. /tissue for more even stimulation. However, the fabrication process of the microfluidic device is time-consuming, and the actuation of the elastic barrier requires the cooperation of an external fluid control device.

To sum up, for methods that utilize microstructures (micropillars, phase guidance, etc.) to generate capillary forces and temporary/virtual barrier-guided gels, the existence of varying degrees in tissue culture efficiency, manipulation difficulty, barrier construction, etc., is unavoidable. However, there is an urgent need for innovative solutions to improve gel patterning to realize cell/tissue culture on microfluidic platforms.

SUMMARY OF THE INVENTION

In view of the defects in the prior art, the purpose of the present invention is to provide a microfluidic organ chip with a soluble temporary barrier and a preparation method thereof.

According to one aspect of the present invention, there is provided a microfluidic organ chip with a soluble temporary barrier, comprising:

A glass sheet substrate to provide support;

The PDMS microfluidic chip layer on the glass substrate, the PDMS microfluidic chip layer includes a central tissue chamber and a first culture fluid microchannel and a second microfluidic channel on both sides of the central tissue chamber. Culture fluid microchannel, the central tissue chamber is used to perfuse hydrogel;

A soluble temporary barrier located on the glass sheet substrate, the soluble temporary barrier is formed of water-soluble PVA, and the soluble temporary barrier includes a microfluidic channel arranged in the first culture medium and the central tissue cavity A first barrier between chambers and a second barrier between the second culture fluid microchannel and the central tissue chamber; the soluble temporary barrier acts as a guide barrier for hydrogel perfusion, in PVA After dissolving, unobstructed culture medium perfusion is performed into the corresponding microfluidic channel.

According to another aspect of the present invention, there is provided a preparation method of the above-mentioned microfluidic organ chip with a soluble temporary barrier, the method comprising:

providing a glass flake base, and closely adhering the screen printing mask on the glass flake base;

completely filling the mesh of the screen printing mask with the PVA solution;

After the PVA solution is cured, the screen printing mask is separated from the glass sheet substrate to form a soluble temporary barrier firmly fixed on the surface of the glass sheet substrate;

A PDMS microfluidic chip layer is provided, and the patterned side of the PDMS microfluidic chip layer is aligned with the soluble temporary barrier and then bonded to the glass sheet substrate to obtain a microfluidic organ with a soluble temporary barrier chip.

Compared with the prior art, the present invention has at least one of the following beneficial effects:

1. The present invention proposes the use of non-toxic, water-soluble PVA powder as the material for making the temporary barrier. Experiments have found that the powder can be dissolved in water to form a viscous liquid, and can be completely dissolved in water again after solidification. Printing technology is used as a patterned fabrication method for temporary barriers; in the gel perfusion experiments of organ chips, the soluble temporary barrier structure can effectively guide the path of the gel and dissolve completely after perfusion of the medium. The organ chip of the present invention not only completes the patterning of the hydrogel, but also achieves barrier-free culture fluid perfusion after dissolving.

3. Compared with the traditional physical barrier design, the present invention can give more sufficient and uniform fluid stimulation to the extracellular matrix. In the vascularization experiment, the growth of blood vessels in the chip proves that the organ chip has a microphysiological environment more in line with cell/tissue culture. In angiogenesis experiments, sprouted branches would invade the ECM along any location where the soluble temporary barrier dissolves, which is more suitable for angiogenesis than the gap between two adjacent micropillars in a micropillar array. The organ chip of the present invention is more conducive to realizing the construction of a human-like microphysiological environment, can provide sufficient fluid stimulation, and is conducive to realizing barrier-free contact during multi-tissue co-cultivation. The soluble temporary barrier design of the present invention is compatible with different organ chips.

Description of drawings

Other features, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments with reference to the following drawings:

1 is a schematic flowchart of a method for preparing a microfluidic organ chip with a soluble temporary barrier according to an embodiment of the present invention;

2 is a schematic structural diagram of a PDMS microfluidic chip layer in an embodiment of the present invention;

3 is a schematic diagram of DTB solubility and hydrogel patterning test in an embodiment of the present invention;

4 is a schematic diagram of blood vessel growth in a single-chamber DTB microfluidic organ chip according to an embodiment of the present invention;

5 is a schematic diagram of angiogenesis of a medium-sized dual-chamber DTB microfluidic organ chip according to an embodiment of the present invention;

6 is a schematic diagram of the blood vessel growth on the sixth day of the non-isometric dual-chamber DTB microfluidic organ chip and the isometric dual-chamber DTB microfluidic organ chip in an embodiment of the present invention;

7 is a schematic diagram of the ability of DTB to confine hydrogel patterning under a non-linear structure in an embodiment of the present invention;

FIG. 8 is a schematic diagram of performing confocal imaging and particle perfusion on the generated vascular network according to an embodiment of the present invention;

Among them, in the figure: 1 is PVA solution, 2 is screen printing mask, 3 is glass substrate, 4 is scraper, 5 is soluble temporary barrier, 6 is PDMS microfluidic chip layer, 7 is hydrogel, 8 is the culture fluid microchannel, R1 is the _first culture fluid microchannel, R2 is the central tissue chamber, _R3 is the _second culture fluid microchannel, V1 is the _first culture fluid loading port, _V2 The second medium loading port, _V3 is the third medium loading port, _V4 is the fourth medium loading port, _V5 is the first hydrogel loading port, and _V6 is the first hydrogel loading port.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention. In the description of the embodiments of the present invention, it should be noted that the terms "first" and "second" in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It is to be understood that the data so used may be interchanged under appropriate circumstances such that the embodiments of the invention described herein can be practiced in sequences other than those illustrated or described herein.

In the description of the embodiments of the present invention, DTB, DTB structure, soluble temporary barrier, and soluble temporary barrier all represent the same meaning, ECM stands for extracellular matrix, PDMS stands for polydimethylsiloxane, PVA stands for polyvinyl alcohol, and DTB Organ chip, DTB microfluidic organ chip, organ chip with soluble temporary barrier, and microfluidic organ chip with soluble temporary barrier have the same meaning, and microfluidic channel and culture fluid microfluidic channel have the same meaning.

An embodiment of the present invention provides a microfluidic organ chip with a soluble temporary barrier for patterning extracellular matrix (ECM, Extracellular Matrix) of the organ chip. Referring to FIG. 1, the organ chip includes: a glass for providing support A sheet substrate 3, a PDMS (polydimethylsiloxane) microfluidic chip layer and a dissolvable temporary barrier 5 (DTB, Dissolvable Temporary Barrier) on the glass sheet substrate 3; wherein, the PDMS microfluidic chip layer 6 includes The central tissue chamber R ₂ and the first culture fluid microchannel R ₁ and the second culture fluid micro channel R ₃ located on both sides of the central tissue chamber R ₂ , the central tissue chamber R ₂ is used for perfusion hydrogel The soluble temporary barrier 5 is formed of water-soluble PVA (polyvinyl alcohol), and the soluble temporary barrier ₅ includes a first barrier and a device arranged between the _first culture fluid microchannel R1 and the central tissue chamber R2. The _second barrier between the second culture fluid microfluidic channel _R3 and the central tissue chamber R2; the soluble temporary barrier 5 acts as a guiding barrier for hydrogel perfusion, and proceeds to the corresponding microfluidic channel after the PVA is dissolved. Barrier-free culture fluid perfusion, that is, after the first barrier is dissolved, the first culture fluid microchannel R ₁ is subjected to barrier-free culture fluid perfusion, and after the second barrier is dissolved, barrier-free culture fluid perfusion is performed into the second culture fluid microchannel R ₃ . Culture fluid perfusion.

The structure of the soluble temporary barrier 5 matches the structure of the PDMS microfluidic chip layer 6. In some specific embodiments, the first barrier and the second barrier are both elongated, and the first barrier is located in the microfluidic layer of the first culture solution. Inside the flow channel R1, the second barrier is located inside the second culture fluid microchannel _R3 , the distance between the _first barrier and the _second barrier is 100-150 μm larger _than the width W2 of the central tissue chamber R2 to limit The ECM forms an ideal patterning effect within the central tissue chamber R2, and does not affect the bonding of the PDMS microfluidic chip layer 6 and the glass substrate 3, which not only ensures the PDMS microfluidic chip layer 6 and the glass substrate 3. success rate, and improve the success rate of hydrogel infusion.

In some specific embodiments, the length of the first barrier is greater than the width of the opening at the intersection of the central tissue chamber R2 and the _first culture fluid microchannel R1, and the length of the _second barrier is greater _than the length of the central tissue chamber R2 and the first culture fluid microchannel R1. The width of the opening at the intersection of the _two culture fluid microchannels _R3 ; preferably, the width of the opening at the intersection of the central tissue chamber R2 and the _first culture fluid microchannel R1 is the same as the width of the opening at the intersection of the central tissue chamber R2 and the _second culture fluid channel R1 The opening width at the intersection of the liquid microfluidic channel _R3 is the same, as shown by W1 in _Fig . 2; the lengths of the first barrier and the second barrier are both larger _than W1 to prevent the hydrogel from leaking to the microfluidics on both sides within the channel.

In some specific embodiments, the height of the first barrier is less than the height of the _first culture fluid microchannel R1, and the height of the second barrier is less than the height of the second culture fluid microchannel _R3 , so as to confine the ECM to the central tissue An ideal patterning effect is formed within the range of the chamber R ₂ , and the bonding between the PDMS microfluidic chip layer 6 and the glass substrate 3 is not affected. Preferably, the heights of the first culture fluid microchannel R ₁ , the second culture fluid microchannel R ₃ and the central tissue chamber R ₂ are 180-200 μm; the heights of the first barrier and the second barrier are 100-120 μm.

In some specific embodiments, the width of the first barrier is smaller than the width of the _first culture fluid microchannel R1, the width of the second barrier is smaller than the width of the second culture fluid microchannel _R3 , preferably, the first barrier And the width of the second barrier is designed to be 100-200 μm, and the widths of the _first culture fluid microchannel R1 and the second culture fluid microchannel _R3 are designed to be 350-500 μm.

In some 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, which can be divided into single or Multiple tissue chambers.

In the microfluidic organ chip with a soluble temporary barrier in the above-mentioned embodiments of the present invention, the soluble temporary barrier can realize stable and leak-free ECM perfusion, which can realize not only single-chamber or multi-chamber, but also regular or irregular Regular ECM patterning, as shown in Figure 3 and Figure 7, the shape of the soluble temporary barrier is a straight line or a curve, which can be set according to actual needs. In addition, the DTB microfluidic organ chip is more conducive to the realization of a human-like microphysiological environment on the chip, which can provide more sufficient and stable fluid stimulation, and is compatible with different organ chip designs. By constructing 3D vascularized microtissues based on different vascularization mechanisms on a DTB microfluidic organ-on-chip, the robustness and flexibility of a soluble temporary barrier for ECM patterning, as well as more closely mimicking human intercellular space flow stimulation, are validated effectiveness.

An embodiment of the present invention also provides a method for preparing the above-mentioned microfluidic organ chip with a soluble temporary barrier. Continuing to refer to FIG. 1 , the method includes:

S1, providing a glass sheet substrate 3, and closely sticking the screen printing mask 2 on the glass sheet substrate 3;

S2. Completely fill the mesh of the screen printing mask 2 with the PVA solution 1;

S3, after the PVA solution 1 is cured, the screen printing mask 2 is separated from the glass sheet substrate 3 to form a soluble temporary barrier 5 that is firmly fixed on the surface of the glass sheet substrate 3;

S4. Provide the PDMS microfluidic chip layer 6, and align the patterned side of the PDMS microfluidic chip layer 6 with the soluble temporary barrier 5 under a microscope and bond it to the glass substrate 3 to obtain a microfluidic chip with a soluble temporary barrier. Fluidic organ-on-a-chip.

In some specific embodiments, step S1 includes: micro-machining the stainless steel screen printing template into a hollow structure matching the shape of the dissolvable temporary barrier 5 by laser cutting, such as an elongated hollow structure, to form a screen printing mask film 2, place the magnet under the glass substrate 3, and stick the screen printing mask 2 tightly on the glass substrate 3 to prevent PVA leakage during screen printing.

PVA solution 1 can be divided into low viscosity, medium viscosity and high viscosity according to viscosity, and PVA solution 1 with higher viscosity is suitable for screen printing process. In some specific embodiments, step S2 includes:

S21. Slowly pour the PVA powder into the distilled water. The mass ratio of the PVA powder to the distilled water is 1:5-1:8. After stirring evenly, use the plastic wrap to seal it. After the bubbles in the solution are dissolved, a viscosity of 44-56 mPa·s is formed. PVA solution 1;

S22, place the PVA solution 1 on the screen printing mask 2, and move the squeegee 4 on the screen printing mask 2 to fill the opening of the screen printing mask 2 with the PVA solution 1, repeat this operation until the screen printing The openings on mask 2 are completely filled.

In some specific embodiments, step S3 includes:

S31, under the condition that the glass sheet substrate 3 and the screen printing mask 2 are kept in close contact, the two are put into an oven at 55-75° C. for 5-10 minutes to cure the PVA solution 1;

S32 , removing the magnet and gently separating the screen printing template 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 above-mentioned embodiment has developed a production process of a soluble temporary barrier based on screen printing PVA material, and proposed a patterned production method using screen printing technology as a temporary barrier, using non-toxic, water-soluble PVA powder as the The material for making the temporary barrier is simple and convenient. Unlike traditional designs, which do not contain a physical barrier like a micropillar array to separate the tissue chamber and the microfluidic channel, there are larger openings between the central tissue chamber _R2 and the culture fluid microfluidic channel, which not only realizes The DTB structure effectively guides the path of the hydrogel, and after the perfusion culture medium completely dissolves PVA, the contact area between the hydrogel and the side culture medium in the tissue chamber will be greatly increased, which is beneficial to the nutrient supply in the subsequent cell culture.

Referring to FIG. 2 , the PDMS microfluidic chip layer 6 in FIG. 1 includes a central tissue chamber R ₂ and a first hydrogel loading port V ₅ and a second hydrogel loading port communicating with the central tissue chamber R ₂ . V ₆ ; the first culture fluid microchannel R1 and the first culture fluid loading port V ₁ and the second culture fluid loading port V ₂ communicating with the first culture fluid microchannel R _{1 ;} the second culture fluid microchannel R ₃ and a third culture solution loading port V ₃ and a fourth culture solution loading port V ₄ communicated with the second culture solution microchannel R ₃ . In some specific embodiments, the PDMS microfluidic chip layer 6 is provided in step S4, wherein the PDMS microfluidic chip layer 6 is prepared by a standard photolithography process, and the preparation method of the PDMS microfluidic chip layer 6 includes:

S41. Prepare a mask: design a mask so that the soluble temporary barrier matches the structure size of the PDMS pattern;

S42. Silicon wafer cleaning: Clean the single-throw silicon wafer in acetone solution, ethanol solution and deionized water in sequence until the polished surface of the silicon wafer is clean and free of stains, use a nitrogen gun to dry the cleaned silicon wafer, and place it on the Bake on a hot plate until moisture is removed;

S43. Gluing and pre-baking: spin-coat the photoresist on the entire surface of the silicon wafer and place it in a glue-spreading machine to homogenize, and then wait for the temperature step to drop to room temperature after pre-baking treatment;

S44, photolithography and post-baking: exposing the photoresist to ultraviolet light through a mask, and then performing post-baking treatment;

S45, development and silanization: take out the silicon wafer cooled to room temperature, immerse it in the developing solution in the yellow light area, then put the silicon wafer into isopropanol solution to remove the developing solution, and then rinse it with deionized water; The gun will blow dry, followed by silanization surface treatment;

S46, pouring the PDMS microfluidic chip layer: the PDMS prepolymer and the curing agent are mixed and poured onto the surface of the photoresist, and the curing treatment is performed, and the PDMS microfluidic chip layer 6 is obtained after demolding and punching operations.

Preferably, the preparation method of PDMS microfluidic chip layer 6 includes:

S41. Prepare a mask: use AutoCAD to design a PDMS pattern structure that matches the size of the soluble temporary barrier structure. The structure of the PDMS microfluidic chip layer 6 is shown in Figure 2, and the shape and size of the two structures can be customized as needed. Set the culture fluid microchannels R ₁ , R ₃ , the tissue chamber R ₂ , the hydrogel loading ports V ₅ , V ₆ , and the culture fluid loading ports V ₁ , V ₂ , V ₃ , V ₄ in the reticle Make the mask transparent and set the rest of the area to black.

S42, silicon wafer cleaning: use an ultrasonic cleaning machine to clean a single-throw silicon wafer in acetone solution, ethanol solution and deionized water in sequence for 8 minutes, until the polished surface of the silicon wafer is clean and free of stains. The cleaned wafers were blown dry using a nitrogen gun and baked on a hot plate at 180°C until moisture was removed.

S43. Gluing and pre-baking: spin-coat SU-8 photoresist on the entire surface of the silicon wafer and place it in a glue homogenizer to obtain SU-8 photoresist with a thickness of 180-200 μm, which is placed in an oven for 60 ℃ for 10 min, then bake at 95 ℃ for 4 h, and finally wait for the temperature step to drop to room temperature.

S44, photolithography and post-baking: expose the photoresist to ultraviolet light through a mask to photolithography the SU-8 adhesive, and place it in an oven at 95° C. for 30 minutes.

S45. Development and silanization: Take out the silicon wafer cooled to room temperature, immerse it in the developing solution for 5-8 minutes in the yellow light area, then put the silicon wafer into isopropanol solution to remove the developing solution, and then rinse it with deionized water . And blow dry with a nitrogen gun, and then carry out silanization surface treatment.

S46. Inverted PDMS microfluidic chip layer: mix the PDMS prepolymer and its curing agent in a ratio of 10:1 and pour it onto the surface of the SU-8 photoresist, put it in a 70°C oven for 2 hours to cure, and finally cut the PDMS Obtain the required PDMS microfluidic chip.

Continuing to refer to FIG. 1 , in some preferred embodiments, a method for preparing a microfluidic organ chip with a soluble temporary barrier includes:

S1. As shown in (a) in Figure 1, a stainless steel plate with a thickness of 120 μm is micro-machined into a strip-shaped hollow structure by laser cutting as a screen printing mask 2, and is closely attached to the glass substrate 3 with a magnet , to prevent liquid leakage during screen printing.

S2. As shown in (b) of FIG. 1 , the PVA solution 1 with high viscosity is placed on the screen printing mask 2 , and the scraper 4 is moved to fill the opening mesh with the PVA solution 1 . This process can be repeated several times until the mesh of the screen printing mask 2 is completely filled, and then the whole is placed in an oven at 60° C. for 7 min. In this step, PVA powder can be divided into low viscosity, medium viscosity and high viscosity according to the viscosity. Pour an appropriate amount of medium-viscosity PVA powder into distilled water slowly, the mass ratio of PVA to distilled water is 1:5, and after stirring evenly, seal it with plastic wrap. After the bubbles in the solution are dissolved, it can be used for screen printing, and the obtained PVA solution has a viscosity of 1 Higher, suitable for screen printing process.

S3. As shown in (c) of FIG. 1 , after the PVA is cured, remove the magnet and then separate the screen printing mask 2 from the glass substrate 3 , and the soluble temporary barrier 5 can be firmly fixed on the surface of the glass substrate 3 .

S4. As shown in (d) in FIG. 1, oxygen plasma surface treatment is performed on the PDMS microfluidic chip layer 6 and the glass substrate 3, and the patterned side of the PDMS microfluidic chip layer 6 is aligned with the temporary dissolution barrier 5. Bond with glass substrate 3, and place the bonded chip on a hot plate at 152°C for 10 minutes to enhance the bonding effect between the two.

As shown in (e) of Figure 1, the design of the gap between the two temporary barriers should be slightly larger than the width of the tissue chamber R ₂ in Figure 2 to prevent the hydrogel from leaking into the side culture fluid microchannel R ₁ , R ₃ . In addition, in order to facilitate better alignment during bonding, the length of the dissolving temporary barrier 5 should be designed to be larger than the width of the opening of the tissue chamber R ₂ .

In the preparation method of the microfluidic organ chip with a soluble temporary barrier in the above-mentioned embodiments of the present invention, the soluble material is patterned on a glass sheet substrate by a screen printing process, and after the soluble material is cured, the glass sheet substrate is Bonding with polydimethylsiloxane (PDMS) microfluidic chip to prepare DTB microfluidic organ chip. Compared with the conventional method of patterning ECM using physical barriers such as micro-pillar arrays in traditional microfluidic organ chips, the embodiment of the present invention can realize unobstructed fluid perfusion and increase the effective contact area between ECM and fluid, which will directly determine The uniformity and strength of cell/tissue stimulation by fluid within the ECM, and the integrity of the basement membrane at the ECM interface. Since the present invention can solve the key technical problems faced by the existing general hydrogel patterning methods, especially for closed microfluidic chips, it is expected to be able to develop novel in vitro organ chip models and other microfluidic-based cell/chip models. A new paradigm for tissue culture analysis.

Referring to Fig. 3, in order to verify the feasibility of a soluble temporary barrier design for organ chip extracellular matrix patterning and its preparation method proposed by the present invention, DTB solubility and hydrogel patterning tests were carried out, including The following steps:

S100, as shown in (a) and (b) in Figure 3, respectively corresponding to the cross-sectional schematic diagram of the chip after the screen printing step and the bonding step, (e) and (f) in Figure 3 are the enlarged physical images under the microscope . After the glass substrate 3 is bonded to the PDMS microfluidic chip layer 6 , the first culture fluid microchannel R ₁ and the second culture fluid microchannel R ₃ are clearly visible, and are not blocked by the soluble temporary barrier 5 . Perfusion is also not affected. Here, even if the thickness of the soluble temporary barrier 5 is half the height of the tissue chamber, the hydrogel 7 can smoothly travel along the soluble temporary barrier ₅ and be firmly confined within the central tissue chamber R2 without The first culture fluid microchannel R ₁ and the second culture fluid microchannel R ₃ will overflow to the side against the meniscus pinning effect.

S200. As shown in FIG. 3(c), the hydrogel 7 mixed with human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (NHLF) is perfused into the central tissue chamber R ₂ , because the hydrogel With high water content, the soluble temporary barrier 5 will be partially dissolved after being in close contact with it for 1 min, and the actual effect is shown in (g) in Figure 3. Since the soluble temporary barrier 5 dissolves for a longer length of time than the time required for the polymerization of the hydrogel ₇ , it does not affect the hydrogel patterning within the central tissue cavity R2.

S300, as shown in (d) in Figure 3, after the hydrogel 7 is solidified, the cell culture fluid is perfused into the culture fluid microchannel 8, and the soluble temporary barrier 5 is completely dissolved after 1 min, realizing the realization of ECM and culture fluid. There is no barrier-free contact between them, and the actual effect is shown in (h) in Figure 3.

Therefore, in addition to leak-free stable ECM patterning, this design provides more adequate nutrient supply and fluid stimulation to cultured cells/tissues. The DTB structure dissolves spontaneously in a liquid environment without the need for specific chemical reactions or the intervention of external equipment.

The actual culture effects of microfluidic organ chips with different structures are described below to demonstrate the feasibility of the design of soluble temporary barriers for extracellular matrix patterning of organ chips and their preparation methods.

Referring to (a)-(h) in Figure 4, it is the pictures of blood vessel growth in the single-chamber DTB microfluidic organ chip from day 1 to day 13, and the structure corresponds to the chip in (e)-(h) in Figure 3 structure. Among them, (a) to (d) in Figure 4 are bright field microscope pictures, and (e) to (h) in Figure 4 are fluorescence microscopy pictures, and the scale bar in the figure is 500 μm. The tissue chamber was filled with hydrogel mixed with HUVEC and NHLF, and the initial seeding concentration of both was 5×10 ⁶ /mL.

Referring to Figure 4, the width of the central tissue chamber R2 is 1 mm, and the width of the culture medium microchannel is 350 μm. Under the fluid stimulation on both sides, it can be seen from the figure that the HUVECs have begun to fragment on the 5th day under the induction of NHLF. Grows and forms connections; lumens begin to form and gradually thicken from day 9, resulting in a 3D capillary network. On the 13th day of growth, the blood vessel density and lumen diameter further increased, and the lumen diameter in the local area could be close to 100 μm, which provided an excellent channel for the delivery of small drug molecules. Due to the large opening between the central tissue chamber R2 and the culture fluid microchannel, the distribution and size of the lumen openings are completely unrestricted, which can better reproduce the capillary network anastomosed in vivo.

Referring to Fig. 5 (a) and (b), it is the images of blood vessel growth on the 0th and 13th days of the equal-sized dual-chamber DTB microfluidic organ chip, and the scale bar in the figure is 500 μm. It can be seen from the figure that, similar to the single-chamber structure, when the blood vessels grow to the 13th day, the capillary network becomes more mature, the lumen size increases, and the vascular structure tends to grow in the direction consistent with the direction of the fluid shear force, that is, the so-called mechanical migration, a key process in vascular remodeling.

Referring to Fig. 6 (a) and (b), the images of blood vessel growth on day 6 of the non-isometric dual-chamber DTB microfluidic organ chip and the isometric dual-chamber DTB microfluidic organ chip, respectively , the scale bar in the figure is 500 μm. It can be seen from the figure that, regardless of the structure, a 3D capillary network has been formed by the 6th day of culture. The results of the angiogenesis experiment performed on the dual-chamber demonstrate the effectiveness of the microfluidic organ chip with a soluble temporary barrier and its preparation method in the design of a multi-chamber structure in the embodiment of the present invention.

Referring to Fig. 7(a)-(d), to further verify the ability of DTB to confine hydrogel patterning under non-linear structure, the circular tissue chamber was divided into three adjacent compartments by two DTBs. . (a) and (b) in FIG. 7 are bright-field microscope pictures, and (c) and (d) in FIG. 7 are fluorescence microscope pictures. The scale bar in the figure is 500 μm. 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 realized the patterning of the hydrogel, as shown in Fig. 7(c). After the DTB was dissolved, the hydrogel mixed with human brain astrocytoma cells (U87MG) was injected into the central oval tissue chamber. The network migrated in a radial arrangement, as shown in Fig. 7(b) and (d). This experimental result demonstrates that tumor cells with highly metastatic properties are able to migrate against gradients of growth factors and nutrients. This tissue compartment can interact with the ECM in a non-physical barrier manner to enable the construction of a vascularized tumor platform. In addition, the precise spatial separation of one tissue chamber through the DTB structure can form three chambers to achieve the co-culture of three-dimensional microvessels on both sides and tumor cells to simulate the growth of human tumor tissue.

To further verify whether the formed microvascular network is three-dimensional and to demonstrate the perfusability of the microvascular network, confocal imaging and particle perfusion were performed on the generated vascular network. Referring to Fig. 8 (a) and (b), confocal microscopy fluorescence imaging demonstrated three-dimensional distribution of blood vessels at different heights within a thickness of 200 μm, in addition, horizontal (XY plane) and vertical (XZ plane and YZ plane) cross-sections The confocal images of the formed microvessels have hollow tubes with a diameter of about 50 μm.

Referring to Figure 8 (c), fluorescent microparticles with a diameter of 5 μm were introduced into the microfluidic channel of one side of the culture medium with high hydrostatic pressure through the medium loading port, and the fluorescent microparticles quickly and easily passed through the microvascular network and entered the other side. microfluidic channels without non-ideal leakage.

To assess the non-physiological leakage properties of the generated 3D microvessels, 70 kDa FITC-dextran with green fluorescence was also perfused into the vessel lumen. Referring to Figure 8 (d), as time goes by, the 3D microvascular network gradually filled with dextran flows from the microchannel with high pressure on one side to the microvascular area with low pressure on the other side. After 10 minutes of perfusion, No dextran was observed outside the vascular lumen. These experimental results all prove that the microvascular network formed in the DTB organ chip has excellent transport performance of small molecules, and the tube wall has a strong barrier performance. It provides a good platform for future in vitro studies of intravascular drug delivery and cancer targeted therapy.

The above experiments can prove that the above embodiments of the present invention propose a patterned fabrication method using screen printing technology as a temporary barrier, and use non-toxic, water-soluble PVA powder as a fabrication material for the temporary barrier. Experiments have found that the powder can be dissolved in water to form a viscous liquid, and can be completely dissolved in water again after solidification, which is suitable for the screen printing process. At the same time, in the gel perfusion experiment of DTB organ chip, the DTB structure can effectively guide the path of the gel and completely dissolve after perfusion of the medium. The DTB Organ Chip not only completed the patterning of the hydrogel, but also achieved barrier-free culture fluid perfusion after dissolution. Therefore, compared with the traditional physical barrier design, more adequate and uniform fluid stimulation can be given to the extracellular matrix. In the vascularization experiment, the growth of blood vessels in the chip proved that the DTB organ chip has a microphysiological environment more in line with cell/tissue culture. In angiogenesis experiments, sprouting branches invade the ECM along any location where DTB dissolves, and the gap between two adjacent micropillars in a micropillar array is more suitable for angiogenesis. The DTB organ chip is more conducive to the construction of a human-like microphysiological environment, can provide sufficient fluid stimulation, and is conducive to achieving barrier-free contact during multi-tissue co-culture. The DTB design is compatible with different organ chips. The microfluidic organ chip of the above embodiment provides innovative application value in the field of biomedical engineering, especially for related researches on the construction of an in vitro vascularized tumor disease model and the screening of new anti-tumor drugs.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above-mentioned specific embodiments, and those skilled in the art can make various variations or modifications within the scope of the claims, which do not affect the essential content of the present invention. The above-mentioned preferred features can be used in any combination as long as they do not conflict with each other.

Claims (10)

1. a microfluidic organ chip with a soluble temporary barrier is characterized in that, comprising: A glass sheet substrate to provide support; The PDMS microfluidic chip layer on the glass substrate, the PDMS microfluidic chip layer includes a central tissue chamber and a first culture fluid microchannel and a second microfluidic channel on both sides of the central tissue chamber. Culture fluid microchannel, the central tissue chamber is used to perfuse hydrogel; A soluble temporary barrier located on the glass sheet substrate, the soluble temporary barrier is formed of water-soluble PVA, and the soluble temporary barrier includes a microfluidic channel arranged in the first culture medium and the central tissue cavity A first barrier between chambers and a second barrier between the second culture fluid microchannel and the central tissue chamber; the soluble temporary barrier acts as a guide barrier for hydrogel perfusion, in PVA After dissolving, unobstructed culture medium perfusion is performed into the corresponding microfluidic channel. 2 . The microfluidic organ chip with a soluble temporary barrier according to claim 1 , wherein the first barrier is located inside the first culture fluid microfluidic channel, and the second barrier is located in the Inside the second culture fluid microchannel, the distance between the first barrier and the second barrier is slightly larger than the 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 intersection of the central tissue chamber and the first culture fluid microchannel. 4 . The length of the second barrier is greater than the width of the opening at the intersection of the central tissue chamber and the second culture fluid microfluidic channel to prevent the hydrogel from leaking into the microfluidic channels on both sides . 4 . The microfluidic organ chip with a soluble temporary barrier according to claim 1 , wherein the height of the first barrier is less than the height of the first culture fluid microfluidic channel, and the second barrier The height of the second culture fluid microchannel is smaller than the height of the second culture fluid microchannel, the width of the first barrier is smaller than the width of the first culture fluid microchannel, and the width of the second barrier is smaller than that of the second culture fluid microchannel. The width of the runner. 5. The microfluidic organ chip with a soluble temporary barrier according to claim 4, wherein the first culture fluid microchannel, the second culture fluid microchannel and the central tissue cavity The height of the chamber is 180-200 μm; the heights of the first and second barriers are 100-120 μm; the widths of the first and second barriers are designed to be 100-200 μm, and the first culture The width of the liquid micro-channel and the second culture fluid micro-channel is designed to be 350-500 μm. 6. The preparation method of the microfluidic organ chip with a soluble temporary barrier according to any one of claims 1-5, characterized in that, comprising: providing a glass flake base, and closely adhering the screen printing mask on the glass flake base; completely filling the mesh of the screen printing mask with the PVA solution; After the PVA solution is cured, the screen printing mask is separated from the glass sheet substrate to form a soluble temporary barrier firmly fixed on the surface of the glass sheet substrate; A PDMS microfluidic chip layer is provided, and the patterned side of the PDMS microfluidic chip layer is aligned with the soluble temporary barrier and then bonded to the glass sheet substrate to obtain a microfluidic organ with a soluble temporary barrier chip. 7 . The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 6 , wherein the step of closely adhering the screen printing mask on the glass sheet substrate comprises: Laser cutting microfabricates a stainless steel screen printing template into a hollow structure that matches the shape of the soluble temporary barrier to form a screen printing mask, which is tightly attached to the glass sheet substrate with a magnet . 8. The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 6, wherein the fully filling the mesh holes of the screen printing mask with PVA solution comprises: Slowly pour the PVA powder into the distilled water, the mass ratio of the PVA powder to the distilled water is 1:5-1:8, and after stirring evenly, seal it with plastic wrap, and after the bubbles in the solution are dissolved, a PVA solution with a preset viscosity is formed; Place the PVA solution on the screen printing mask and move a squeegee over the screen printing mask to fill the openings of the screen printing mask with the PVA solution, repeating this operation until the screen printing The openings in the mask are completely filled. 9 . The method for preparing a microfluidic organ chip with a soluble temporary barrier according to claim 7 , wherein, after the PVA solution is cured, the screen printing mask and the glass sheet substrate are applied. 10 . separation, including: Under the condition that the glass sheet substrate and the screen printing mask are kept in close contact, the two are put into an oven at 55-75° C. for 5-10 minutes to cure the PVA solution; The magnet was removed and the screen-printed stencil was gently separated from the glass sheet substrate, leaving a soluble temporary barrier on the glass sheet substrate surface. 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 adopts a standard Photolithography process preparation, the preparation method of the PDMS microfluidic chip layer includes: Preparing the reticle: Design the reticle so that the soluble temporary barrier matches the structure size of the PDMS pattern; Wafer cleaning: Clean single-throw silicon wafers in acetone solution, ethanol solution and deionized water in sequence until the polished surface of the wafer is clean and free of stains. Use a nitrogen gun to dry the cleaned wafers and place them on a hot plate. Bake until moisture is removed; Gluing and pre-baking: spin-coat the photoresist on the entire surface of the silicon wafer and place it in a glue-spreading machine to homogenize, and then wait for the temperature step to drop to room temperature after pre-baking; Photolithography and post-baking: Expose the photoresist to ultraviolet light through a mask, and then perform post-baking treatment; Development and silanization: Take out the silicon wafer cooled to room temperature, immerse it in the developing solution in the yellow light area, then put the silicon wafer into isopropanol solution to remove the developing solution, and rinse it with deionized water; Blow dry, followed by silanization surface treatment; Inverted PDMS microfluidic chip layer: the PDMS prepolymer and its curing agent are mixed and poured onto the surface of the photoresist, and then cured, and the PDMS microfluidic chip layer is obtained after demolding and drilling operations.

Images