Method for constructing biomimetic capillary network, blood separation method and application of three-dimensional microtissue perfusion culture

Abstract

The application provides a construction method of a bionic capillary network, a blood separation method and application of three-dimensional microtissue perfusion culture, and relates to the technical field of biomaterials and bionics. The construction method comprises the following steps: preparing a fishbone-shaped microchannel die on a silicon wafer substrate; spin-coating an elastic polymer solution on the surface of the die to solidify and form a flexible film, and bonding the flexible film with a flat packaging film; performing patterned cutting on the side wall to cut out a lateral opening, so as to obtain a two-dimensional microchannel network structure; pre-stretching the elastic stretching substrate along at least one direction, bonding at least part of the nodes of the two-dimensional microchannel network structure to the surface of the elastic stretching substrate, and driving the nodes to occur bending deformation to form a three-dimensional bionic capillary network by using the retraction force. The bionic capillary network has a microchannel design with a fishbone-shaped lateral opening, highly imitates the structure of in-vivo capillary vessels, and is suitable for the fields of separation and treatment of trace blood or perfusion culture of three-dimensional microtissues.

Description

Technical Field

[0001] This invention relates to the fields of biomaterials and bionics, and in particular to a method for constructing a bionic capillary network, a blood separation method, and its application in three-dimensional microtissue perfusion culture. Background Technology

[0002] In the human circulatory system, capillaries, as tiny blood vessels distributed at the very end of the vascular network, are typically less than 10 micrometers in diameter, about one-tenth the diameter of a human hair. Although imperceptible to the naked eye, these tiny blood vessels undertake the most crucial physiological task of the circulatory system—mediating the exchange of substances between blood and surrounding tissue cells. This unique function is achieved through the extremely thin structure of the capillary wall (composed of only a single layer of endothelial cells) and the countless tiny pores distributed along the wall. It is this structure, possessing both "thinness" and "leakage," that makes it a "cargo distribution station" connecting arteries, veins, and tissue cells, ensuring that oxygen and nutrients can cross the vessel wall to reach the cells, while simultaneously assisting in the removal of metabolic waste into the bloodstream.

[0003] It is noteworthy that the "leakage" or permeability characteristics of capillaries in different organs and tissues vary significantly, primarily due to differences in the size of the micropores in the capillary walls. For example, the capillaries (i.e., hepatic sinusoids) in the liver are wider and more tortuous, with larger pores distributed on the endothelial cells, and even an incomplete basement membrane. This high permeability allows large plasma molecules and even blood cells to directly contact hepatocytes, thus efficiently performing physiological functions such as detoxification and metabolism. In contrast, the capillaries in the glomeruli of the kidneys possess a special filtration barrier that effectively blocks the leakage of red blood cells and large protein molecules, while allowing water and small to medium-sized solutes to filter out and form primary urine. In the brain, capillaries form the well-known blood-brain barrier, with tightly connected endothelial cells that almost completely prevent the free diffusion of substances, allowing only lipid-soluble small molecules or those transported by specific proteins to cross, thus constructing a tight protective barrier for the central nervous system. These capillary networks with differentiated opening structures in the human body are fundamental to maintaining the normal physiological functions of various organs.

[0004] However, in the field of biomedical research, traditional two-dimensional cell culture models and animal experiments have long faced challenges such as insufficient simulation of the real physiological environment, limited accuracy of experimental results, and low research and development efficiency. Therefore, reconstructing a highly biomimetic physiological microenvironment in vitro has become a key technological path to overcome these bottlenecks, among which the construction of a functional biomimetic capillary network is particularly crucial. By building a perfusionable three-dimensional vascular network, researchers can simulate the in vivo environment, enabling the continuous delivery of nutrients to deep three-dimensional tissues and effectively removing metabolic waste, thereby maintaining the normal metabolic activity of internal cells. For example, organoid chips integrating biomimetic vascular networks can not only more realistically simulate in vivo tissue structures but can also be widely used in high-throughput drug screening and disease model research, significantly shortening the new drug development cycle. However, most current mainstream three-dimensional vascular network construction technologies rely on 3D printing, which is often time-consuming when dealing with complex and delicate structures and is difficult to integrate efficiently and with living cells with low damage.

[0005] Meanwhile, the development of biomimetic capillary structures also has profound application value in the fields of clinical diagnostics and medical testing. Blood testing, as a routine and crucial means of assessing a patient's condition and diagnosing diseases, typically requires separating the collected blood to obtain serum or blood cell sediment for subsequent analysis. This process traditionally relies on special blood collection tubes and centrifuges, which is not only cumbersome but also requires large blood sample volumes. For patients who are weak, prone to fainting at the sight of blood, or young children, venipuncture to collect large amounts of blood not only causes significant pain but also increases the risk of infection. Although modern testing technology has advanced to the point where reliable results can be obtained with only extremely small sample volumes, clinical blood collection still cannot escape the dependence on large blood volumes due to limitations in traditional centrifugation techniques. In particular, the emerging microfluidic detection technology, whose core advantage lies in micro-volume and rapid detection, could greatly promote the development of point-of-care testing by developing a biomimetic technology that can directly separate serum from minute amounts of blood without centrifugation, providing strong technical support for modern precision medicine. Summary of the Invention

[0006] To address the technical problems existing in the prior art, embodiments of the present invention provide a method for constructing a biomimetic capillary network, a blood separation method, and its application in three-dimensional microtissue perfusion culture. The technical solution is as follows:

[0007] This invention provides a method for constructing a biomimetic capillary network, comprising the following steps:

[0008] Fishbone-shaped microchannel protrusions were fabricated on a silicon wafer substrate using photolithography.

[0009] An elastic polymer solution is spin-coated onto the surface of the fishbone-shaped microchannel punch and cured to form a flexible film with a fishbone-shaped microchannel pattern. The flexible film is then bonded to a flat encapsulation film to form a closed fishbone-shaped microchannel.

[0010] A patterned cutting process is used to cut along the sidewall of the closed fishbone-shaped microfluidic channel using laser processing technology, and multiple spaced lateral openings are cut into the sidewall to form a biomimetic fishbone-shaped microfluidic channel that communicates with the outside, thereby obtaining a two-dimensional microfluidic network structure with the biomimetic fishbone-shaped microfluidic channel.

[0011] The elastic stretching substrate is pre-stretched in at least one direction, and at least some nodes of the two-dimensional microfluidic network structure are bonded to the surface of the pre-stretched elastic stretching substrate. The pre-stretching stress of the elastic stretching substrate is released, and the retraction force of the elastic stretching substrate is used to drive the two-dimensional microfluidic network structure to buckle and deform to form a three-dimensional biomimetic capillary network.

[0012] Optionally, in step S1, the specific process of fabricating the fishbone-shaped microchannel punch on the silicon wafer substrate using photolithography includes:

[0013] The silicon wafer substrate undergoes pretreatment including cleaning and drying.

[0014] Prepare a mask template with a fishbone-shaped microchannel pattern;

[0015] Photoresist is spin-coated onto the surface of the pretreated silicon wafer substrate and pre-baked to remove the solvent;

[0016] After aligning the photomask with the silicon wafer substrate, it is placed on the photoresist for exposure; the exposed photoresist is then baked, and a developer is used to dissolve and remove specific exposed areas, forming a fishbone-shaped microchannel pattern on the photoresist.

[0017] The photoresist is hardened; then, using an etching machine, the hardened photoresist is used as a masking layer to etch, resulting in a fishbone-shaped microchannel punch with a preset height.

[0018] Optionally, the elastic polymer is selected from one or more of polydimethylsiloxane, styrene-ethylene / butene-styrene block copolymer, polydioxanone, or thermoplastic polyurethane.

[0019] Optionally, the biomimetic fishbone-shaped microfluidic channel includes a main channel and multiple branch channels, the multiple branch channels being spaced apart on both sides of the main channel, and one end of each of the multiple branch channels being connected to the main channel, while the other end forming the lateral opening.

[0020] Optionally, the main channel is provided with an injection inlet and an injection outlet.

[0021] Optionally, the lateral opening is rectangular; the width of the lateral opening is 1-5 μm and the height is 5-25 μm; the length of the branch channel is 10-100 μm and the spacing between adjacent branch channels is 10-100 μm.

[0022] Optionally, the thickness of both the flexible film and the encapsulation film is 20-100 μm.

[0023] The present invention also provides a biomimetic capillary network obtained by the construction method described above.

[0024] This invention also provides a blood separation method based on a biomimetic capillary network, comprising the following steps:

[0025] Blood is injected into the biomimetic fishbone-shaped microfluidic channels of the aforementioned biomimetic capillary network.

[0026] By utilizing the size exclusion effect of the lateral openings of the biomimetic capillary network, serum in the blood can seep out through the lateral openings, while blood cells are trapped in the biomimetic fishbone-shaped microfluidic channels, thereby achieving the separation of serum and blood cells.

[0027] The present invention also provides an application of a biomimetic capillary network in three-dimensional microtissue perfusion culture, characterized in that cells and hydrogel complex are perfused and solidified in the biomimetic fishbone-shaped microfluidic channels of the above-mentioned biomimetic capillary network, and the solidified cells and hydrogel complex are provided with nutrients by perfusing culture medium into the biomimetic fishbone-shaped microfluidic channels.

[0028] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0029] (1) The biomimetic capillary network constructed in this invention has a microchannel design with fishbone-shaped side openings, which is highly biomimetic to the structure of capillaries in the body. By precisely controlling the size of the side openings, selective control of the permeability of different substances can be achieved; at the same time, the miniaturized size of the microchannels makes it particularly suitable for the separation and processing of small amounts of blood.

[0030] (2) The present invention uses micro-nano processing technology (such as photolithography) to prepare a fishbone-shaped side opening structure, which can greatly ensure the dimensional accuracy and structural uniformity of the microchannel, and realize high-precision control of the cavity size and side opening size.

[0031] (3) The present invention utilizes a tensile elastic substrate to mechanically guide the three-dimensional self-assembly of a two-dimensional network structure. This method retains the high-precision advantage of two-dimensional planar processing and realizes the rapid prototyping of complex three-dimensional microfluidic structures, overcoming the shortcomings of traditional 3D printing technology in constructing fine structures.

[0032] (4) The perfusionable three-dimensional microchannel constructed in this invention can be combined with cells / hydrogels to realize the perfusion culture of cells in the three-dimensional hydrogel. Functionally, it successfully mimics the material exchange and nutrient delivery function of capillaries in the body.

[0033] (5) The perfusionable capillary network can support long-term in vitro perfusion culture of centimeter-level three-dimensional tissues, providing a technical platform for constructing tissue models that are closer to the real in vivo environment, and can be widely used in disease model research, drug screening and tissue development process research.

[0034] (6) This method has good universality. By adjusting the two-dimensional network design and assembly parameters, three-dimensional biomimetic vascular networks with different topologies can be constructed to meet the needs of different application scenarios. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram of the biomimetic fishbone-shaped microfluidic channel of the biomimetic capillary network provided in an embodiment of the present invention;

[0037] Figure 2 This is a schematic diagram of the lateral opening of the biomimetic fishbone-shaped microfluidic channel provided in an embodiment of the present invention;

[0038] Figure 3 This is a diagram of a three-dimensional biomimetic vascular network structure assembled using a tensile elastic substrate, as provided by an embodiment of the present invention.

[0039] Figure 4 This is another embodiment of the present invention, showing a three-dimensional biomimetic vascular network structure assembled using a tensile elastic substrate;

[0040] Figure 5 These are photos of sheep blood diluted 100x passing through a biomimetic capillary network, showing the entrance and exit points (the bottom photo shows the entrance, and the top photo shows the exit).

[0041] Figure 6 The results show the characterization of HeLa cells cultured in assembled three-dimensional biomimetic capillaries for 21 days. (Figure (a) is a light slide micrograph of the cultured three-dimensional whole; (b) is a light slide micrograph of a local PDMS microchannel; (c) and (d) are scanning electron micrographs of cells inside the cultured three-dimensional tissue.) Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0043] Example 1

[0044] This invention provides a method for constructing a biomimetic capillary network, comprising the following steps:

[0045] a. First, the two-dimensional pattern with a fishbone-shaped biomimetic vascular network is designed and obtained by photolithography to form a fishbone-shaped microchannel punch structure;

[0046] The specific process of fabricating a fishbone-shaped microchannel punch with a micron structure on a silicon substrate using photolithography includes: 1) fabricating a microchannel mask for the punch using an MLA150 laser direct writing device; 2) spin-coating a negative photoresist onto a clean silicon wafer surface with a thickness of 10 μm, then placing the mask on the silicon wafer for exposure and using a photolithography machine to transfer the pattern; 3) after removing excess photoresist, placing the silicon wafer in a deep silicon etching machine for deep silicon etching to obtain a punch with an etching depth of 15 μm.

[0047] b. Using magnetron sputtering, a 10 nm thick polytetrafluoroethylene film is sputtered on the surface of the silicon wafer punch as a hydrophobic layer. The silicon wafer punch can be reused to prepare a mold with a flow channel structure.

[0048] c. A PDMS pre-curing solution (10:1 ratio of crosslinking agent) is spin-coated onto the surface of a hydrophobically treated silicon wafer at a speed of 1000 rad / min, and then baked at 120℃ for 3 min to obtain a PDMS film with a microchannel structure; at the same time, PDMS is spin-coated onto a flat silicon wafer surface at a speed of 1500 rad / min and cured to obtain a flat film; finally, the PDMS film with microchannels and the flat film are bonded and sealed by plasma treatment to form a closed fishbone-shaped microchannel.

[0049] d. Using a high-precision picosecond laser cutting device, patterned cuts are made along the sidewall of the closed fishbone-shaped microfluidic channel to cut multiple spaced lateral openings on the sidewall. This process exposes the opening structure on the side of the microfluidic channel to form a biomimetic fishbone-shaped microfluidic channel that communicates with the outside, thereby obtaining a two-dimensional microfluidic network structure with the biomimetic fishbone-shaped microfluidic channel.

[0050] e. Finally, the bonding sites of the two-dimensional microfluidic network structure are bonded to the stretched elastic substrate: a 2 mm thick highly elastic, skin-like silicone elastic substrate is fixed on a stretching table capable of stretching in both the X and Y directions, and stretched by 100% in both directions; then, the bonding sites of the two-dimensional microfluidic network structure are bonded to the stretched highly elastic, skin-like silicone elastic substrate; finally, the stretched elastic substrate is released from the stretching table, thus obtaining a three-dimensional assembled biomimetic capillary network (e.g., Figure 3 (As shown).

[0051] Example 2

[0052] This invention provides a method for constructing a biomimetic capillary network, comprising the following steps:

[0053] a. First, the two-dimensional pattern with a fishbone-shaped biomimetic vascular network is used to obtain the microchannel convex structure through photolithography.

[0054] The specific process of fabricating a fishbone-shaped microchannel punch with a micron structure on a silicon substrate using photolithography includes: 1) fabricating a microchannel mask for the punch using an MLA150 laser direct writing device; 2) spin-coating a negative photoresist onto a clean silicon wafer surface with a thickness of 10 μm, and then placing the mask on the silicon wafer and using a photolithography machine to transfer the pattern; 3) after removing excess photoresist, placing the silicon wafer in a deep silicon etching machine for deep silicon etching to obtain a punch with an etching depth of 15 μm.

[0055] b. After sputtering a 10 nm thick polytetrafluoroethylene film as a hydrophobic layer on the surface of the silicon wafer punch using magnetron sputtering, the silicon wafer punch can be reused to prepare a mold with a flow channel structure.

[0056] c. A 20% wt solution of SEBS in n-hexane is spin-coated onto the surface of a hydrophobically treated silicon wafer at a speed of 800 rad / min, and then dried overnight in a fume hood to obtain a SEBS film; simultaneously, a 20% wt solution of SEBS in n-hexane is spin-coated onto a flat silicon wafer surface at a speed of 800 rad / min and dried to obtain a flat film; finally, the SEBS film with microchannels is thermally bonded to the flat film to form a closed fishbone-shaped microchannel.

[0057] d. Using a high-precision picosecond laser cutting device, patterned cuts are made along the sidewall of the closed fishbone-shaped microfluidic channel to cut multiple spaced lateral openings on the sidewall. This process exposes the opening structure on the side of the microfluidic channel to form a biomimetic fishbone-shaped microfluidic channel that communicates with the outside, thereby obtaining a two-dimensional microfluidic network structure with the biomimetic fishbone-shaped microfluidic channel.

[0058] e. Finally, the bonding sites of the two-dimensional microfluidic network structure are bonded to the stretched elastic substrate: a 2 mm thick highly elastic, skin-like silicone elastic substrate is fixed on a stretching table capable of stretching in both the X and Y directions, and stretched by 100% in both directions; then, the bonding sites of the two-dimensional microfluidic network structure are bonded to the stretched Dragonskin elastic substrate; finally, the stretched elastic substrate is released from the stretching table, thus obtaining a three-dimensional assembled biomimetic capillary network (e.g., ...). Figure 4 (As shown).

[0059] Example 3: Micro-blood separation method

[0060] This invention provides a method for micro-blood separation based on a biomimetic capillary network, comprising the following steps: Purchasing anticoagulant treated sheep blood is diluted 100-fold with physiological saline to prepare a mixture. This mixture is then injected through the main channel of the biomimetic capillary network using a syringe. During this process, blood cells do not pass through the lateral openings of the biomimetic fishbone-shaped microfluidic channel and remain within the channel, while water and other substances can pass through. Ultimately, it can be observed that the blood cell concentration is lower and the flow rate is faster at the inlet, while the blood cell concentration is higher and the flow rate is slower at the outlet. The processed blood volume is 100 microliters, and the results are as follows... Figure 5 As shown.

[0061] Example 4: Three-dimensional cell and tissue culture

[0062] This invention provides an application of a biomimetic capillary network in three-dimensional microtissue perfusion culture, the application method comprising: […]. Figure 3 The perfusion port of the prepared biomimetic vascular network is connected to an injection pump as a perfusion source for the culture medium. The assembled three-dimensional biomimetic vascular network is then placed in a groove, and 10... 8 A suspension of HeLa cells and Matrigel gel at a specific concentration was poured into a biomimetic capillary network and solidified in a 37°C incubator. Perfusion culture in a medium (DMEM high glucose, 10% serum, 1% penicillin-streptomycin) at a rate of 5 μL / min was then performed to support the in vitro formation of three-dimensional tissues. After 21 days of culture, the three-dimensional tissues were removed, fixed with 4% paraformaldehyde, stained, and photographed. Cell characterization results within the three-dimensional tissues are as follows: Figure 6 As shown.

[0063] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing a biomimetic capillary network, characterized in that, Includes the following steps: Fishbone-shaped microchannel protrusions were fabricated on a silicon wafer substrate using photolithography. An elastic polymer solution is spin-coated onto the surface of the fishbone-shaped microchannel punch and cured to form a flexible film with a fishbone-shaped microchannel pattern. The flexible film is then bonded to a flat encapsulation film to form a closed fishbone-shaped microchannel. A patterned cutting process is used to cut along the sidewall of the closed fishbone-shaped microfluidic channel using laser processing technology, and multiple spaced lateral openings are cut into the sidewall to form a biomimetic fishbone-shaped microfluidic channel that communicates with the outside, thereby obtaining a two-dimensional microfluidic network structure with the biomimetic fishbone-shaped microfluidic channel. The elastic stretching substrate is pre-stretched in at least one direction, and at least some nodes of the two-dimensional microfluidic network structure are bonded to the surface of the pre-stretched elastic stretching substrate. The pre-stretching stress of the elastic stretching substrate is released, and the retraction force of the elastic stretching substrate is used to drive the two-dimensional microfluidic network structure to buckle and deform to form a three-dimensional biomimetic capillary network.

2. The construction method according to claim 1, characterized in that, In step S1, the specific process of fabricating a fishbone-shaped microchannel punch on a silicon wafer substrate using photolithography includes: The silicon wafer substrate undergoes pretreatment including cleaning and drying. Prepare a mask template with a fishbone-shaped microchannel pattern; Photoresist is spin-coated onto the surface of the pretreated silicon wafer substrate and pre-baked to remove the solvent; After aligning the photomask with the silicon wafer substrate, it is placed on the photoresist for exposure; the exposed photoresist is then baked, and a developer is used to dissolve and remove specific exposed areas, forming a fishbone-shaped microchannel pattern on the photoresist. The photoresist is hardened; then, using an etching machine, the hardened photoresist is used as a masking layer to etch, resulting in a fishbone-shaped microchannel punch with a preset height.

3. The construction method according to claim 1, characterized in that, The elastic polymer is selected from one or more of polydimethylsiloxane, styrene-ethylene / butene-styrene block copolymer, polydioxanone, or thermoplastic polyurethane.

4. The construction method according to claim 1, characterized in that, The biomimetic fishbone-shaped microfluidic channel includes a main channel and multiple branch channels. The multiple branch channels are spaced apart on both sides of the main channel, and one end of each branch channel is connected to the main channel, while the other end forms the lateral opening.

5. The construction method according to claim 4, characterized in that, The main channel is equipped with an injection inlet and an injection outlet.

6. The construction method according to claim 4, characterized in that, The lateral opening is rectangular; the width of the lateral opening is 1-5 μm and the height is 5-25 μm; the length of the branch channel is 10-100 μm and the spacing between adjacent branch channels is 10-100 μm.

7. The construction method according to claim 1, characterized in that, The thickness of both the flexible film and the encapsulation film is 20-100 μm.

8. A biomimetic capillary network obtained by the construction method according to any one of claims 1-7.

9. A blood separation method based on a biomimetic capillary network, characterized in that, Includes the following steps: Blood is injected into the biomimetic fishbone-shaped microfluidic channels of the biomimetic capillary network as described in claim 8. By utilizing the size exclusion effect of the lateral openings of the biomimetic capillary network, serum in the blood can seep out through the lateral openings, while blood cells are trapped in the biomimetic fishbone-shaped microfluidic channels, thereby achieving the separation of serum and blood cells.

10. An application of a biomimetic capillary network in three-dimensional microtissue perfusion culture, characterized in that, The cells and hydrogel complex are infused and solidified in the biomimetic fishbone-shaped microfluidic channels of the biomimetic capillary network of claim 8, and the solidified cells and hydrogel complex are nutrient-rich by infusing culture medium into the biomimetic fishbone-shaped microfluidic channels.

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