Acellular matrix modified bionic micro-fluidic organ chip constructed by 3D printing composite near-field direct writing and preparation method and application of bionic micro-fluidic organ chip

Abstract

The invention belongs to the technical field of biomedical engineering and tissue engineering, and particularly relates to an acellular matrix modified bionic micro-fluidic organ chip constructed by 3D printing composite near-field direct writing as well as a preparation method and application of the bionic micro-fluidic organ chip. According to the bionic micro-fluidic organ chip prepared by the invention, the microstructure and physiological similarity of a model are remarkably improved, the problems of insufficient bionic property, single cell and the like at present are solved, and the preparation method is simple, is suitable for batch production and solves the problem of high production cost. The invention provides a new technical direction for the construction of the bionic organ model.

Description

Technical Field

[0001] The present invention belongs to the technical fields of biomedical engineering and tissue engineering, and particularly relates to a biomimetic microfluidic organ chip modified by decellularized matrix constructed by 3D printing combined with near-field direct writing, and a preparation method and application thereof. The biomimetic organ chip constructed by the near-field direct writing 3D printing technology, the decellularized matrix modification technology and the microfluidic technology of the present invention is particularly suitable for urethral biomimicry, and is used for in vitro simulating a urethral stricture pathological model and drug screening. Background Art

[0002] Urethral stricture, as a thorny problem in urology, is a great challenge for doctors. Due to the special structure of the urethral tissue and the unique environment of urine, once the urethra is damaged, urine extravasation is very likely to occur, resulting in the submucosal tissue of the urethra being exposed to urine for a long time. And urine is filled with various chemical components, and these components will continuously stimulate the urethra and its surrounding tissues, causing inflammation. Therefore, patients with urethral injury often cannot avoid the occurrence of urethral scar hyperplasia, and the hyperplasia of the scar will gradually block the urethral lumen, thus forming urethral stricture. In recent years, the incidence of urethral stricture has been showing an upward trend in both developed and developing countries, and one person in every three hundred is affected by this disease on average. Urethral stricture not only seriously interferes with the normal urination function of patients, greatly reducing their quality of life, but also long-term urethral stricture may cause life-threatening complications such as renal failure.

[0003] At present, clinically, vascularized tissues (such as scrotal and penile flaps) or free mucosa (such as oral and lingual mucosa) are often used to treat anterior urethral stricture, and end-to-end anastomosis is used to treat posterior urethral stricture. Among many medical units, endoscopic treatment methods such as internal urethrotomy are widely used because of their convenience, especially in the treatment of bulbar urethral stricture, membranous urethral stricture and bladder neck contracture. However, the recurrence problem of urethral stricture has always been a difficult problem faced clinically. According to domestic and foreign research and the actual experience of this center, the recurrence rate after urethral reconstruction is about 20%, and the recurrence rate of endoscopic treatment is even as high as more than 50%. Therefore, whether it is open surgery or endoscopic surgery, how to effectively inhibit the recurrence of urethral stricture and improve the treatment success rate are common challenges. Although there have been some attempts at present, such as methods of local injection of glucocorticoids into scars, etc., to try to reduce the recurrence of stricture after endoscopic treatment, the effect is still not satisfactory. Therefore, it is particularly urgent and important to deeply explore the pathogenesis of urethral stricture and find new prevention and repair treatment strategies.

[0004] The research on the pathological mechanism of urethral stricture has been carried out since the 1970s. Singh's research has shown through histological analysis of animal models and clinical human specimens that when urethral stricture occurs with significant urinary extravasation, fibroblasts in the diseased tissue will increase significantly, and at the same time, the collagen component and collagen type will change significantly. Subsequent studies have also found that this change in collagen component is mainly a significant shift in the content and ratio of type I / III collagen components. The currently more mainstream view also holds that under normal circumstances, the fine regulation of collagen in the extracellular matrix is in a balanced state of continuous synthesis and degradation. Once this balance is broken, the type of collagen changes, and the structure of the protein molecule changes, resulting in changes in the deposition of extracellular matrix collagen and the collagen network structure, that is, extracellular matrix remodeling. Extracellular matrix remodeling can further lead to tissue ischemia, exacerbating the imbalance of collagen synthesis and degradation, thus forming a vicious cycle.

[0005] Although there have been some studies on scars, it is not difficult to see several deficiencies in the current basic research related to urethral scars. 1) The dynamic hydrodynamic model suitable for the urethra is imperfect, so it has not been able to fully fit the hydrodynamic changes and the pathological processes of related factors accompanied by luminal stenosis after urethral scar formation. 2) Current studies are all based on single cells in a two-dimensional plane, but in three-dimensional space, what kind of interactions fibroblasts will have with other surrounding cells, such as epithelial cells, endothelial cells, and smooth muscle cells, and what final changes will occur are still unknown.

[0006] Traditional cell culture and animal model systems play a crucial role in mechanism research and preclinical drug testing. However, traditional cell culture lacks complexity, and animal research is time-consuming, which limits its ability to predict clinical responses and increases the high cost of drug discovery and development. Microphysiological systems (MPS), including organ-on-chip (OOC) technology, are defined as complex, multicellular (currently multi-organ) in vitro culture systems to mimic the physiological characteristics of specific organs or tissues. Some key MPS studies have expanded our understanding of biological systems for disease treatment and prevention and provided hope for OOC technology, filling the gap between in vitro experiments and animal research. Commercial organ-on-chips only provide the most basic configuration and do not achieve the simulation of the microstructure, active factors, and hydrodynamic properties of urethral tissue, so they are not yet suitable for the research of urethral diseases. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the present invention provides a bionic microfluidic organ chip modified by decellularized matrix constructed by 3D printing composite near-field direct writing, and its preparation method and application. Through implementation verification, the bionic microfluidic organ chip of the present invention is used for organ bionics of urethral stricture diseases, and solves the problems of insufficient current bionics, single cells, and high production cost of the urethral stricture organ chip in vitro model. The preparation process of the organ chip provided by the present invention is simple and suitable for mass production. Since the added decellularized matrix is crosslinked with gelatin onto the 3D-printed electrospun membrane, the core biocompatibility of the organ chip is greatly increased, and the two types of cells are separated into two independent culture chambers, making the structure of the organ chip more bionic.

[0008] The present invention provides a diaphragm, which contains a near-field direct writing electrospun membrane and high-temperature extruded fibers attached to the near-field direct writing electrospun membrane and distributed in a grid pattern. A mixture of decellularized matrix and gelatin is uniformly crosslinked on the near-field direct writing electrospun membrane and the high-temperature extruded fibers.

[0009] The raw materials used for the near-field direct writing electrospun membrane and the high-temperature extruded fibers are bioengineering materials with good biodegradability, biocompatibility, non-toxicity, mechanical properties, and processing properties, including but not limited to polycaprolactone PCL, polylactic acid PLA, polyglycolic acid PGA, polytetrafluoroethylene, poly(lactide), chitosan, collagen, alginate, hyaluronic acid, chitosan, agarose, hydroxyapatite HA, β-tricalcium phosphate β-TCP, etc., and further preferably polycaprolactone PCL.

[0010] The preparation of the near-field direct writing electrospun membrane and the high-temperature extruded fibers attached to the near-field direct writing electrospun membrane and distributed in a grid pattern includes: using 3D printing drawing software to draw a model, and slicing to design the sequence and number of layers of near-field direct writing electrospinning and high-temperature extrusion. Further, after slicing, there are a total of five layers. The first layer is used for near-field direct writing electrospinning to construct the near-field direct writing electrospun membrane, and the last four layers are used for high-temperature extrusion to construct high-temperature extruded fibers distributed in a grid pattern.

[0011] The parameters of the near-field direct writing electrospinning include: parameters of the near-field direct writing print head: temperature 80°C, voltage 4 kV, and platform height 3.5 mm.

[0012] The preparation of the high-temperature extruded fibers includes: parameters of the high-temperature extrusion print head: temperature 70°C, air pressure 500 kPa.

[0013] In the mixture of decellularized matrix and gelatin, the mass ratio of decellularized matrix to gelatin is 1:50 - 100, preferably 1:75.

[0014] The cross-linking step of the mixture of the decellularized matrix and gelatin includes: soaking the near-field direct-write electrospun membrane and the high-temperature extruded fibers distributed in a grid pattern attached to the near-field direct-write electrospun membrane in the decellularized matrix composite solution, taking them out and drying, then soaking them in a cross-linking agent for cross-linking, and taking them out and drying.

[0015] The preparation of the decellularized matrix composite solution includes: mixing the decellularized matrix solution and the gelatin solution to obtain the decellularized matrix composite solution. In the decellularized matrix composite solution, the mass ratio of the decellularized matrix to gelatin is 1:50 - 100, preferably 1:75; the concentration of the decellularized matrix is 3.5 - 5 mg / mL, preferably 4 mg / mL.

[0016] The preparation of the decellularized matrix solution includes: taking the decellularized matrix powder and collagenase and placing them in a hydrochloric acid solution, digesting them at a constant temperature to obtain a digestive solution, and then adding a sodium hydroxide solution and a buffer solution to the digestive solution for neutralization to obtain the decellularized matrix solution. Further, a buffer solution can be added for dilution after neutralization. The decellularized matrix powder is bladder decellularized matrix powder, further rabbit bladder decellularized matrix powder.

[0017] The preparation of the decellularized matrix powder includes: grinding the freeze-dried tissue to obtain the decellularized matrix powder. Further, grinding the freeze-dried bladder tissue to obtain the bladder decellularized matrix powder.

[0018] In the hydrochloric acid solution, the mass ratio of the bladder decellularized matrix powder to collagenase is 8 - 13:1, preferably 10:1; the concentration of the bladder decellularized matrix powder is 8 - 13 mg / mL, preferably 10 mg / mL. The concentration of the hydrochloric acid solution is 0.005 - 0.02 M, preferably 0.01 M.

[0019] The temperature of the constant-temperature digestion is 25 ± 5 °C, and the time is 48 ± 5 h; further, it is a 48-hour constant-temperature digestion at 25 °C. During the constant-temperature digestion process, the hydrochloric acid solution is in a flowing state. Further, the constant-temperature digestion is carried out on a shaker.

[0020] The volume ratio of the sodium hydroxide solution to the digestive solution is 1:9 - 11, preferably 1:10; the concentration of the sodium hydroxide solution is 0.05 - 0.2 M, preferably 0.1 M.

[0021] The volume ratio of the buffer solution to the digestive solution is 1:8 - 10, preferably 1:9; the buffer solution includes but is not limited to borate buffer solution, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution, N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer solution, acetic acid-acetate buffer solution, phosphate buffer solution, etc., and further preferably phosphate buffer solution (10×).

[0022] The gelatin solution is a 10% - 25% gelatin solution, preferably a 15% gelatin solution.

[0023] The cross-linking agent used for cross-linking is an ethanol solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 9-11:1, preferably 10:1; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 4.5-6 mg / mL, preferably 5 mg / mL. The ethanol solution is ethanol with a content of more than 90%, further 90%-98% ethanol, and even further 90% ethanol.

[0024] The present invention also provides a preparation method for the above-mentioned membrane sheet, and the steps include:

[0025] (1) Preparing a near-field direct-write electrospun membrane and high-temperature extruded fibers distributed in a grid pattern attached to the near-field direct-write electrospun membrane by 3D printing to obtain a 3D printed membrane sheet;

[0026] (2) Cross-linking a mixture of decellularized matrix and gelatin on the 3D printed membrane sheet.

[0027] In step (1), the preparation of the near-field direct-write electrospun membrane and the high-temperature extruded fibers distributed in a grid pattern attached to the near-field direct-write electrospun membrane includes: drawing a model with drawing software, and designing the sequence and number of layers of near-field direct-write electrospinning and high-temperature extrusion by 3D printing slicing. Further, there are a total of five layers after slicing. The first layer performs near-field direct-write electrospinning to construct the near-field direct-write electrospun membrane, and the last four layers perform high-temperature extrusion to construct the high-temperature extruded fibers distributed in a grid pattern.

[0028] The parameters of the near-field direct-write electrospinning include: near-field direct-write print head parameters: temperature 80°C, voltage 4 kV, and platform height 3.5 mm.

[0029] The preparation of the high-temperature extruded fibers includes: high-temperature extrusion print head parameters: temperature 70°C, air pressure 500 kPa.

[0030] In step (1), the 3D printed membrane sheet is soaked in 75% alcohol overnight, then repeatedly washed with deionized water, and finally dried at 30°C.

[0031] In step (2), the preparation of the mixture of decellularized matrix and gelatin cross-linked on the 3D printed membrane sheet includes: soaking the 3D printed membrane sheet in a decellularized matrix composite solution, taking it out and drying it, then soaking it in a cross-linking agent for cross-linking, and taking it out and drying it.

[0032] The preparation of the acellular matrix composite solution includes: mixing the acellular matrix solution with the gelatin solution to obtain the acellular matrix composite solution. In the acellular matrix composite solution, the mass ratio of the acellular matrix to gelatin is 1:50 - 100, preferably 1:75; the concentration of the acellular matrix is 3.5 - 5 mg / mL, preferably 4 mg / mL.

[0033] The preparation of the acellular matrix solution includes: taking the acellular matrix powder and collagenase and placing them in a hydrochloric acid solution, digesting at a constant temperature to obtain a digestion solution, and then adding a sodium hydroxide solution and a buffer solution to the digestion solution for neutralization to obtain the acellular matrix solution. Further, a buffer solution can be added for dilution after neutralization. The acellular matrix powder is a bladder acellular matrix powder, further a rabbit bladder acellular matrix powder.

[0034] The preparation of the acellular matrix powder includes: grinding the freeze-dried tissue to obtain the acellular matrix powder. Further, grinding the freeze-dried bladder tissue to obtain the bladder acellular matrix powder.

[0035] In the hydrochloric acid solution, the mass ratio of the acellular matrix powder to collagenase is 8 - 13:1, preferably 10:1; the concentration of the acellular matrix powder is 8 - 13 mg / mL, preferably 10 mg / mL. The concentration of the hydrochloric acid solution is 0.005 - 0.02 M, preferably 0.01 M.

[0036] The temperature of the constant temperature digestion is 25 ± 5 °C, and the time is 48 ± 5 h; further, it is a constant temperature digestion at 25 °C for 48 h. During the constant temperature digestion process, the hydrochloric acid solution is in a flowing state. Further, the constant temperature digestion is carried out on a shaker.

[0037] The volume ratio of the sodium hydroxide solution to the digestion solution is 1:9 - 11, preferably 1:10; the concentration of the sodium hydroxide solution is 0.05 - 0.2 M, preferably 0.1 M.

[0038] The volume ratio of the buffer solution to the digestion solution is 1:8 - 10, preferably 1:9; the buffer solution includes but is not limited to borate buffer solution, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution, N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid buffer solution, acetic acid-acetate buffer solution, phosphate buffer solution, etc., and is further preferably phosphate buffer solution (10×).

[0039] The gelatin solution is a 10% - 25% gelatin solution, preferably a 15% gelatin solution.

[0040] The crosslinking agent used for crosslinking is an ethanol solution containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 9-11:1, preferably 10:1; the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 4.5-6 mg / mL, preferably 5 mg / mL. The ethanol solution is ethanol with a content of more than 90%, further 90%-98% ethanol, and even further 90% ethanol.

[0041] The diaphragm provided above in the present invention can be used to prepare products for cell culture and / or products for bionic organs or tissues. The products include but are not limited to materials, devices, chips, or equipment, etc. The materials, devices, chips, or equipment, etc. are applicable to microfluidic systems. Further, the products for bionic organs or tissues include but are not limited to products for bionics of urethral organs or tissues, products for urethral stricture organs or tissues, products for screening drugs for prevention or treatment of urethral stricture, etc.

[0042] The present invention also provides a bionic microfluidic organ chip, which includes a chip housing and a chamber wrapped by the chip housing. The diaphragm of the present invention above is fixed in the chamber. An inlet and an outlet communicating with the chamber are provided on the chip housing; it also includes a microfluidic system, and the chamber is connected to the microfluidic system through the inlet and outlet communicating with it. Further, the diaphragm divides the space of the chamber, and the divided chamber spaces are independent of each other. Inlets and outlets respectively communicating with each divided chamber space are provided on the chip housing, and each divided chamber space is connected to the microfluidic system through the inlet and outlet communicating with it.

[0043] A fastening device is also provided on the chip housing. The fastening device is used to fix the bionic microfluidic organ chip to ensure the airtightness of the bionic microfluidic chip. The fastening device is also provided with channels respectively corresponding to and communicating with the inlets and outlets of each divided chamber space, and an observation window for observing the chamber space. The cleaning of the chamber, the input of the culture solution and cultured cells in the chamber, and the connection between the chamber space and the microfluidic system are carried out through the channels respectively corresponding to and communicating with the inlets and outlets.

[0044] As an implementation manner, the bionic microfluidic organ chip provided by the present invention includes two upper and lower chip housings and a chamber wrapped by the two upper and lower chip housings. A diaphragm is fixed in the chamber. The diaphragm divides the chamber into two chamber spaces. The two divided chamber spaces are independent of each other, and inlets and outlets communicating with the chamber spaces are respectively provided in each chamber space.

[0045] Fastening devices are provided on both chip housings. On the fastening device of each chip housing, there are channels respectively corresponding to and communicating with the inlet and outlet of the corresponding chamber space, and an observation window for observing the corresponding chamber space.

[0046] The present invention also provides a method for preparing a bionic microfluidic organ chip, and the steps include:

[0047] (1) Place the material for forming the chip housing into a 3D-printed chip housing mold, perform curing treatment, and demold to obtain the chip housing;

[0048] (2) Assemble the chip housing obtained in step (1) with the diaphragm prepared above in the present invention to obtain a bionic microfluidic organ chip.

[0049] In step (1), the material for forming the chip housing includes, but is not limited to, silicone compounds and curing agents. The mass ratio of silicone compounds to curing agents is 9 - 12:1, preferably 10:1. The silicone compounds include, but are not limited to, any one or any combination of polydimethylsiloxane, cyclomethicone, aminosiloxane, polyether polysiloxane copolymer, and are preferably polydimethylsiloxane. The curing agents include, but are not limited to, methylhydrogensiloxane, hydrogen-containing siloxane, and are preferably methylhydrogensiloxane.

[0050] In step (1), cure at 80 ± 5°C for 2 - 3 hours, preferably cure at 80°C for 2 hours.

[0051] In step (1), before placing the material for forming the chip housing into the 3D-printed chip housing mold, evenly spray a release agent on the surface of the 3D-printed chip housing mold.

[0052] In step (1), the 3D printing process of the chip housing mold includes: drawing a model with drawing software, performing slice design with 3D printing slicing software, and preparing the chip housing mold by stereolithography 3D printing technology. The material of the chip housing mold is photosensitive resin.

[0053] In step (2), a fastening device is further added during the assembly process. The material of the fastening device is polymethyl methacrylate (PMMA).

[0054] In step (2), a microfluidic system is further added during the assembly process. The microfluidic system is connected to the bionic microfluidic organ chip.

[0055] Both the diaphragm provided above in the present invention and the bionic microfluidic organ chip provided above can be used for preparing physiological organ or tissue bionic models, and / or pathological organ or tissue bionic models, and / or drug screening models.

[0056] Among the following products provided by the present invention, they contain the diaphragm provided above in the present invention or the bionic microfluidic organ chip provided above:

[0057] (1) Physiological organ or tissue bionic model;

[0058] (2) Pathological organ or tissue bionic model;

[0059] (3) Drug screening model.

[0060] Furthermore, the above physiological organ or tissue bionic model is a urethral organ or tissue bionic model, the pathological organ or tissue bionic model is a urethral stricture organ or tissue model, and the drug screening model is a urethral stricture prevention or treatment drug screening model.

[0061] The above physiological organ bionic model of the present invention is applicable to the urethral bionic model. The above pathological organ bionic model of the present invention is applicable to the urethral stricture bionic model.

[0062] The advantages of the present invention are as follows:

[0063] In recent years, the emerging near-field direct-write electrospinning technology has become the most promising technology for constructing three-dimensional tissue structures with strict geometric meanings. In our implementation research, we found that by adjusting the process parameters, it can achieve micron-level cell orientation induction, realize the simulation of the structure of normal tissues or organs, and then combined with the application of bladder acellular matrix, it can significantly improve the microstructure and physiological similarity of the tissue model, pointing out a new direction for the field of urethral stricture treatment. A reasonably designed microfluidic bioreactor can maximize the simulation of the hydrodynamic characteristics of the urethra.

[0064] In the implementation research of the present invention, the near-field direct-write electrospinning technology and urinary tract acellular matrix are used to construct a bionic urethral tissue, and the microfluidic technology is used to simulate the in-vivo microenvironment of urethral stricture occurrence from multiple dimensions such as urine mechanical stimulation. The present invention is expected to establish an in-vitro simulation model of urethral stricture and deeply clarify its occurrence mechanism, laying a foundation for establishing specific prevention and treatment strategies for urethral stricture.

[0065] The acellular matrix-modified bionic microfluidic organ chip prepared by 3D printing composite near-field direct writing provided by the present invention and its preparation method provide a new technical direction for constructing organ physiological or pathological models. Description of the Drawings

[0066] Figure 1 It is the size diagram of the organ chip housing mold prepared by 3D printing in Example 1 of the present invention.

[0067] Figure 2 It is the physical diagram of the organ chip housing mold prepared by 3D printing in Example 1 of the present invention.

[0068] Figure 3This is the physical diagram of the organ chip housing after demolding in Embodiment 1 of the present invention (left figure), the mechanical test diagram of the organ chip housing (middle figure), and the stress-strain trend diagram of the organ chip housing (right figure).

[0069] Figure 4 This is the BAM composite solution diagram (a1, a2, a3) in Embodiment 2 of the present invention, and the physical observation diagram (a4, a5) of the BAM composite solution crosslinked to the 3D composite printing film. a1 is the physical diagram of the BAM composite solution, a2 is the low-magnification observation diagram of the BAM composite solution in a 96-well plate, a3 is the high-magnification observation diagram of the BAM composite solution in a 96-well plate, a4 is the low-magnification observation diagram of the BAM composite solution crosslinked to the 3D composite printing film, and a5 is the high-magnification observation diagram of the BAM composite solution crosslinked to the 3D composite printing film.

[0070] Figure 5 This is the top view (upper figure) of the organ chip assembled from the organ chip housing and the membrane in Embodiment 2 of the present invention, and the sectional view taken along the A-A plane of the organ chip assembled from the organ chip housing and the membrane (lower figure). Among them, 1 - organ chip housing, 1-1 upper housing of the organ chip, 1-2 lower housing of the organ chip, 2 - chamber in the organ chip, 2-1 upper chamber in the organ chip, 2-2 lower chamber in the organ chip, 3 - membrane: the membrane divides the chamber in the organ chip into two independent upper and lower chambers, 4 - inlet, 4-1 inlet communicating with the upper chamber, 4-2 inlet communicating with the lower chamber, 5 - outlet, 5-1 outlet communicating with the upper chamber, 5-2 outlet communicating with the lower chamber.

[0071] Figure 6 This is the three-dimensional explosion diagram of the organ chip assembled from the organ chip housing, the membrane and the fastening device in Embodiment 2 of the present invention. 6 - fastening device, 6-1 upper fastening device, 6-2 lower fastening device, 7 - fastening screw, 8 - fastening nut, 9 - pore passage provided on the fastening device and communicating with the inlet, 9-1 pore passage provided on the upper fastening device and communicating with the inlet of the upper chamber, 9-2 pore passage provided on the lower fastening device and communicating with the inlet of the lower chamber, 10 - pore passage provided on the fastening device and communicating with the outlet, 10-1 pore passage provided on the upper fastening device and communicating with the outlet of the upper chamber, 10-2 pore passage provided on the lower fastening device and communicating with the outlet of the lower chamber, 11 - observation window, 11-1 observation window provided on the upper fastening device, 11-2 observation window provided on the lower fastening device.

[0072] Figure 7 This is the top view and the sectional view taken along the D-D plane of the organ chip assembled from the organ chip housing, the membrane and the fastening device in Embodiment 2 of the present invention.

[0073] Figure 8This is a physical diagram of the bionic microfluidic organ chip assembled from the organ chip housing, membrane, fastening device and microfluidic system in Example 2 of the present invention.

[0074] Figure 9 This is a comparison diagram of the induced orientation of cells by the 3D composite printed membrane (the BAM composite solution prepared in the uncrosslinked Example 2) prepared in Example 2 of the present invention. D1, D3, and D5 respectively refer to the culture times of 1, 3, and 5 days.

[0075] Figure 10 This is a spatial distribution result diagram of co-culture using the bionic microfluidic organ chip in Example 3 of the present invention. a is a two-dimensional cell staining image on the side of culturing urothelial cells; b is a two-dimensional cell staining image on the side of culturing fibroblasts, c is a top view of the three-dimensional cell staining image of culturing double-layer cells, and d is a front view of the three-dimensional cell staining image of culturing double-layer cells.

[0076] Figure 11 This is a cell result diagram of adding different intervention factors related to urethral stricture in Example 3 of the present invention. Detailed implementation manners

[0077] The embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present invention rather than all of them. Other embodiments obtained by those skilled in the art without any creative work fall within the protection scope of this patent.

[0078] To describe the present invention more clearly, the bionic microfluidic urethral organ chip provided by the present invention, as well as the preparation method and application, will be described in more detail below with reference to the embodiments.

[0079] Example 1:

[0080] Use SolidWorks 2021 3D software to draw the mold design diagram required for the organ chip. The general size of the mold is 95mm×48mm×6mm, which is divided into 4 arrayed chambers, and the specific dimensions are as Figure 1 shown. Next, import the model into the 3D printing slicing software and use the stereolithography 3D printing technology to prepare the chip mold, with the material being photosensitive resin.

[0081] Mix the main agent and curing agent of polydimethylsiloxane (PDMS) in a ratio of 10:1. During the mixing process, it is necessary to ensure sufficient stirring to achieve a uniform mixing effect. Then, use the method of vacuum degassing to remove the bubbles in the mixture to ensure the quality and performance of the chip. Next, pour the degassed PDMS mixture into the mold prepared by stereolithography printing. To ensure the demolding quality, evenly spray the release agent on the mold surface before adding the material.

[0082] Subsequently, according to the characteristics of the material, the mixed material and the mold were placed in an environment of 80 °C for curing. The curing time was 2 hours. During this process, PDMS would gradually cure to form a stable structure. Finally, a tweezer was used for demolding operation to take out the cured chip from the mold.

[0083] Figure 1 It is the dimensional drawing of the mold for the organ chip housing prepared by 3D printing. Figure 2 It is the physical picture of the mold for the organ chip housing. From Figure 1 and Figure 2 it can be seen that the mold for the organ chip housing prepared by 3D printing has four demolding chambers, and can form 2 groups of chip housings at one time, improving the preparation efficiency. The surface of the mold in the physical picture is flat and smooth, which is suitable for subsequent demolding.

[0084] Figure 3 They are the physical picture of the organ chip housing, the mechanical test chart of the organ chip housing, and the stress-strain trend chart of the organ chip housing. From Figure 3 it can be seen that the prepared organ chip conforms to the conventional mechanical properties of the PDMS material and is applicable to subsequent experiments.

[0085] Example 2:

[0086] Use 3D printing drawing software to draw the diaphragm model. According to the designed size of the organ chip, the size of the diaphragm was designed to be 37.8×7.8×0.4 mm, with a total of five layers after slicing. The first layer was the electrostatic near-field direct writing layer, and the last four layers were the high-temperature extrusion grid layers. At the same time, add the required PCL material to the printing cartridge before printing, and pre-test the process parameters: High-temperature extrusion print head: The temperature is set at 70 °C, and the air pressure is 500 kPa. Near-field direct writing print head: The temperature is set at 80 °C, the voltage is 4 kV, and the platform height is 3.5 mm. Then start composite printing. After 10 minutes, the printing is completed. Turn off the high-voltage power supply, take out the 3D composite printed diaphragm, and observe it under a microscope. If the fibers are evenly distributed, it is qualified. Immerse the printed diaphragm in 75% alcohol overnight to eliminate contamination, then repeatedly wash the membrane with deionized water, and place it in a clean oven at 30 °C to dry overnight.

[0087] The freeze-dried rabbit bladder tissue was placed in a grinder for grinding to obtain bladder acellular matrix (BAM) powder, and then collagenase was added to obtain a bladder acellular matrix solution. 100 mg of BAM powder and 10 mg of collagenase were placed in 10 mL of hydrochloric acid solution (0.01 M), and digested in a constant temperature shaker at 25 °C for 48 h to obtain a BAM digestion solution. Then, NaOH solution (0.1 M) with a volume of 1 / 10 of the digestion solution and 10× phosphate buffer solution (PBS) with a volume of 1 / 9 of the digestion solution were added for neutralization, and finally diluted with PBS to obtain a BAM solution. Then, the BAM solution was added to a 15% gelatin solution to obtain a final BAM composite solution ( Figure 4 in a1, a2, a3), the mass ratio of BAM to gelatin was 1:75, and the concentration of BAM was 4 mg / mL. Then, the prepared 3D composite printing membrane was soaked in the BAM composite solution for 60 s, taken out and dried at 40 °C for 15 min, then soaked in a cross-linking agent for cross-linking for 4 h, taken out and repeatedly rinsed with deionized water for 7 - 10 times, and dried to obtain a membrane ( Figure 4 in a3, a4). More preferably, the solvent of the cross-linking agent was 90% ethanol, and the solutes were 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) at 5 mg / mL and N-hydroxysuccinimide (NHS) at 0.5 mg / mL.

[0088] According to the size of the organ chip, an organ chip fixture was made of PMMA material. Needle fixing holes (the needle fixing holes communicate with the corresponding inlets and outlets) and observation windows were respectively arranged on the upper and lower sides of the fixture for convenient later experiments. Six groups of screws were arranged around the fixture to fix the organ chip housing and the membrane, and the transparent PMMA material was also convenient for experimental observation. According to the size of the cell culture cavity flow channel, a needle with a diameter of 0.8 mm and a silicone hose with an inner diameter of 0.8 mm were selected. After the needle and the silicone hose were made with the fixture, they were soaked in 75% alcohol and irradiated with ultraviolet light overnight to eliminate contamination. After the sterilized organ chip housing and the membrane were assembled with the fixture according to the "sandwich" structure, a micro syringe was used to check its airtightness. If there is no liquid leakage and the flow rate is normal, it is qualified. The qualified organ chip was connected to the microfluidic system, and the physical diagram is shown in Figure 8 .

[0089] The bionic microfluidic organ chip assembled in this example is as shown in Figures 5 - 8As shown in the figure, the upper housing 1-1 and the lower housing 1-2 are assembled into a chip housing 1. Inside the chip housing 1, a chamber 2 is formed for cell culture and imitating physiological or pathological environments. An inlet 4 and an outlet 5 communicating with the chamber 2 are provided on the chip housing 1. The diaphragm 3 is fixed between the upper housing 1-1 and the lower housing 1-2 of the chip housing 1, separating the chamber 2 into an upper chamber 2-1 and a lower chamber 2-2. The upper housing 1-1 is provided with an inlet 4-1 and an outlet 5-1 communicating with the upper chamber 2-1, and the lower housing 1-2 is provided with an inlet 4-2 and an outlet 5-2 communicating with the lower chamber 2-2. An external fastening device 6 including an upper fastening device 6-1 and a lower fastening device 6-2 is provided outside the housing 1. The upper fastening device 6-1 is located on one side of the upper housing 1-1, and the lower fastening device 6-2 is located on one side of the lower housing 1-2. A channel 9-1 communicating with the inlet 4-1 of the upper housing 1-1 is provided on the upper fastening device 6-1, a channel 10-1 communicating with the outlet 5-1 of the upper housing 1-1 is provided on the upper fastening device 6-1, and an observation window 11-1 for observing the upper chamber 2-1 is further provided on the upper fastening device 6-1. A channel 9-2 communicating with the inlet 4-2 of the lower housing 1-2 is provided on the lower fastening device 6-2, a channel 10-2 communicating with the outlet 5-2 of the lower housing 1-2 is provided on the lower fastening device 6-2, and an observation window 11-2 for observing the lower chamber 2-2 is provided on the lower fastening device 6-2. The observation window 11-1 and the observation window 11-2 form an observation window 11 on the fastening device 6. The upper fastening device 6-1 and the lower fastening device 6-2 perform airtight fastening on the upper housing 1-1 and the lower housing 1-2 through 6 groups of fastening screws 7 and fastening nuts 8 provided around. The upper chamber 2-1 is used for cleaning the chamber, inputting and outputting substances such as cells and culture media, and connecting injectants and microfluidic systems through the inlet 4-1, the channel 9-1 communicating with the inlet 4-1, the outlet 5-1, and the channel 10-1 communicating with the outlet 5-1. The lower chamber 2-2 is used for cleaning the chamber, inputting and outputting substances such as cells and culture media, and connecting injectants and microfluidic systems through the inlet 4-2, the channel 9-2 communicating with the inlet 4-2, the outlet 5-2, and the channel 10-2 communicating with the outlet 5-2.

[0090] Figure 4 Figure 4 shows the direct observation of the bladder acellular matrix gelatin solution under the microscope and the physical observation of the cross-linked 3D composite printed film. Through Figure 4 It can be seen that the BAM tissue in the prepared BAM composite solution is evenly distributed (a1, a2, a3) and can be well combined with the 3D composite printed film (a3, a4), providing a basis for cell proliferation.

[0091] Figure 5 Figure 10 shows the top view (upper figure) of the organ chip assembled by the organ chip housing and the diaphragm and the schematic cross-sectional view A-A (lower figure). ThroughFigure 5 It can be seen that the diaphragm divides the organ chip into upper and lower cell culture channels (chambers), and there are inlets and outlets connecting the upper and lower cell culture channels (chambers) respectively, enabling three-dimensional culture of upper and lower double-layer cells.

[0092] Figures 6 - 8 Shown are the three-dimensional exploded view, top view, cross-sectional view, and physical diagram of the bionic microfluidic organ chip assembled from the organ chip housing, diaphragm, and fastening device. Figures 6 - 8 It can be seen that on the basis of the combination of the original organ chip housing and diaphragm, the assembled bionic microfluidic organ chip has added a clamp, a silicone hose, and an injection pump. And through the passage test of two-color liquids, it is proved that the airtightness of the microfluidic system is intact and there is no leakage between them.

[0093] Example 3:

[0094] To promote cell adhesion in the microchannels, the inner surfaces of all microchannels were incubated with gelatin solution for 12 hours and then sterilized.

[0095] To verify the cell-loading ability of the 3D printed composite electrostatic direct writing membrane, in a 24-well plate, an uncrosslinked 3D composite printed membrane (prepared in Example 2) was used as the experimental group, and a blank cell slide was used as the control. Fibroblast HFF-1 was used to explore the proliferation morphology of cells on the membrane, and its morphology was observed by immunofluorescence experiment to judge whether it could achieve the effect of inducing orientation. Specifically, after cells were seeded simultaneously under two sets of conditions, on the 1st, 3rd, and 5th days, 4% paraformaldehyde was added, fixed at room temperature for 15 min, washed 3 times with PBS for 5 min each time, then 0.3% Triton X-100 was added for permeabilization for 20 min, washed 3 times with PBS for 5 min each time, then blocked with 3% BSA for 1 h and washed 3 times with PBS for 5 min each time; after adding the primary antibody and incubating overnight at 4°C, it was washed 3 times with PBS for 5 min each time. Then the secondary antibody was added and incubated at room temperature in the dark for 1 h; after thorough washing with PBS, 1 mL DAPI was added to stain the cell nuclei for 10 min and then thoroughly washed with PBS. The blank slide and the 3D printed composite electrostatic direct writing membrane were respectively placed on a glass slide, covered with a coverslip, observed and photographed under a microscope, fluorescence images were obtained under the same conditions, and randomly selected ones were photographed and analyzed. The results are shown in Figure 9 .

[0096] After sterilizing the assembled organ chip, needles, and silicone hoses, they were placed in a laminar flow hood. After connecting needles at both ends, the microchannels were thoroughly rinsed 3 times with sterile PBS. Then, cell culture-grade collagen was incubated on both sides of the flow channel and placed in an incubator for 8 hours to improve the hydrophilicity of the microchannels and facilitate cell adhesion. After the collagen incubation was completed, the organ chip was taken out, and 1.5×10 6HFF-1 fibroblasts were inoculated and placed in an incubator for 9 hours to allow them to adhere to the wall. After that, the organ chip was taken out of the incubator, inverted to extrude the printing side, and 1.5×10 6 SV-HUC-1 urothelial cells were inoculated. After inoculation, they were also placed statically in the incubator. At the same time, both ends of the electrostatic direct writing side of the cultured fibroblasts were connected to the microfluidic system. The specific operation was as follows: The needle and the silicone hose were connected and simultaneously inserted into the sterilized syringe pump. According to the human urine flow rate, the injection flow rate of the syringe pump was set to 4 uL / min, and the injection volume was 5 mL (the culture medium was changed regularly). The culture medium composition was 90% high-glucose DMEM + 10% fetal bovine serum + 1% penicillin / streptomycin. Similarly, after 9 hours, both ends of the extrusion printing side of the cultured urothelial cells were connected to the microfluidic system, and the flow rate was the same as that of the electrostatic direct writing side. Then, co-culture and immunofluorescence were performed, and the steps were the same as above. The results are shown in Figure 10 .

[0097] Finally, a urethral stricture model was established. The experimental groups were a blank control group without drugs, a group configured with 10 ng / ml TGF-β1, a group with 100 nM rapamycin, and a group with 10 ng / ml TGF-β1 + 100 nM rapamycin. Each group had 5 ml of culture medium, and stenosis was induced by introducing it into the organ chip at the same pump speed. After dynamic perfusion for 48 h, the bladder acellular matrix / PCL membrane could be removed for subsequent immunofluorescence staining, and the steps were the same as above. The results are shown in Figure 11 .

[0098] Figure 9 This is a comparison diagram of the induced orientation of cells by the 3D composite printing membrane prepared in Example 2 of the present invention (the BAM composite solution prepared in the non-crosslinked Example 2). From Figure 9 it can be seen that the control group was a blank coverslip, showing that the proliferation direction of cells was random, while in the electrostatic direct writing group, the cells proliferated regularly according to the filaments of the 3D composite electrostatic direct writing printing membrane, indicating that successful results were obtained for the orientation induction of cells.

[0099] Figure 10 This is a result diagram of the spatial distribution of co-culture using the bionic microfluidic organ chip in Example 3 of the present invention. From Figure 10 the plane distribution diagram and the cross-sectional view, it can be seen that the two types of cells were respectively distributed on the side of the BAM composite gelatin solution (i.e., the high-temperature extrusion grid side) and the 3D electrostatic direct writing printing side, thus presenting different proliferation directions and results, which was in line with the expected results of the invention and realized the co-culture of the two types of cells in space.

[0100] Figure 11 This is a cell result diagram of adding different intervention factors related to urethral stricture in Example 3 of the present invention. Figure 11It is reflected that in the organ-on-a-chip, the cells in the TGF-β group showed fibrotic aggregation characteristics, which is in line with the predicted phenomenon at the cellular level of urethral stricture. Under the co-intervention of TGF-β and rapamycin, this phenomenon was offset, indicating that the invention shows results that conform to objective laws in the research related to urethral stricture and can be used as an in vitro research method to replace animal experiments.

[0101] Examples 1-3 provide an organ-on-a-chip applicable to the research of urethral stricture diseases. The organ-on-a-chip is prepared by the replica molding method and 3D composite printing technology and combined with a biomaterial to form a diaphragm structure with high biocompatibility. The prepared organ-on-a-chip verified its proliferation feasibility through the inoculation of two types of cells, fibroblasts and urothelial cells, and verified the biomimicry of the constructed diaphragm through immunofluorescence biological experiments. At the same time, by adding different intervention factors related to urethral stricture, the potential of the invention for urethral stricture research was demonstrated.

[0102] The above are only the preferred embodiments of the present invention. It should be noted that all embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

Claims

1. A diaphragm, characterized in that: The invention comprises a near-field direct writing electrostatic spinning membrane and high-temperature extruded fibers attached to the near-field direct writing electrostatic spinning membrane and distributed in a grid shape. The near-field direct writing electrostatic spinning membrane and the high-temperature extruded fibers are uniformly cross-linked with a mixture of acellular matrix and gelatin.

2. A diaphragm according to claim 1, characterized in that: The mass ratio of the decellularized matrix to the gelatin in the mixture of the decellularized matrix and gelatin is 1:50-100.

3. The method for preparing a membrane according to claim 1 or 2, characterized in that the steps include: (1) 3D printing to prepare a near-field direct writing electrospinning membrane and high-temperature extruded fibers attached to the near-field direct writing electrospinning membrane in a grid-like distribution, thereby obtaining a 3D printed membrane sheet; (2) Cross-linking a mixture of decellularized matrix and gelatin on a 3D printed membrane.

4. The preparation method according to claim 3, characterized in that: In step (1), a model is drawn using a drawing software, and the sequence and number of layers of near-field direct writing electrospinning and high-temperature extrusion are designed by 3D printing slices to prepare a near-field direct writing electrospinning membrane and high-temperature extruded fibers attached to the near-field direct writing electrospinning membrane in a grid-like distribution.

5. A membrane according to claim 1 or 2 or a membrane prepared by the preparation method according to claim 3 or 4 is used for preparing products for cell culture and / or products for bionic organs or tissues.

6. A bionic microfluidic organ chip, characterized in that: The invention comprises a chip shell and a chamber wrapped by the chip shell, wherein a membrane according to claim 1 or 2 or a membrane prepared by the preparation method according to claim 3 or 4 is fixed in the chamber, and an inlet and an outlet communicating with the chamber are arranged on the chip shell.

7. The bionic microfluidic organ chip according to claim 6, characterized in that: The microfluidic system is also included, and the chamber is connected to the microfluidic system via an inlet and an outlet communicating with the chamber.

8. The method for preparing a bionic microfluidic organ chip according to claim 6 or 7, characterized in that the steps include: (1) placing a chip shell molding material in a 3D printed chip shell mold, performing a curing treatment, and demolding the mold to obtain a chip shell; (2) Assembling the chip shell prepared in step (1) with the membrane prepared above to obtain a bionic microfluidic organ chip.

9. A diaphragm as described in claim 1 or 2, or a diaphragm prepared by the preparation method as described in claim 3 or 4, or a bionic microfluidic organ chip as described in claim 6 or 7, or a bionic microfluidic organ chip prepared by the preparation method as described in claim 8 is used to prepare physiological organ or tissue bionic models, and / or pathological organ or tissue bionic models, and / or drug screening models.

10. At least one of the following products contains a membrane according to claim 1 or 2, or a membrane prepared by the preparation method according to claim 3 or 4, or a bionic microfluidic organ chip according to claim 6 or 7, or a bionic microfluidic organ chip prepared by the preparation method according to claim 8: (1) Bionic models of physiological organs or tissues; (2) Bionic models of pathological organs or tissues; (3) Drug screening model.

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