CN112206074B - Tubular vascular-like structure and construction method thereof - Google Patents

Abstract

The invention provides a tubular tissue-like structure and a construction method thereof. The method comprises the following steps: A. preparing printing ink; B. culturing cells for printing in vitro to obtain a cell culture solution; C. and (3) respectively connecting the printing ink and the cell culture fluid with different spray heads of the biological printer, printing the printing ink and the cell culture fluid onto the winding rod together to form a seamless tubular tissue, and removing the winding rod to obtain the hollow tubular tissue-like structure. The method forms a tubular structure with controllable structure and cell/material composition by a horizontal winding type cell 3D printing technology. The tubular structure is similar to the shape and the size of a human body vessel, has mechanical and biological properties required by a human body, has clinical application potential, and can be used for aspects of medicine detection, tissue engineering, regenerative medicine, in-vitro physiological model/pathological model/pharmacological model construction, tissue/organ/human body chip and the like.

Description

Tubular blood vessel-like structure and construction method thereof

Technical field

The present invention relates to the field of biomedical engineering, and specifically to a tubular blood vessel-like structure and a construction method thereof.

Background technique

The human body's vasculature system is widely distributed, providing transportation of nutrients, oxygen, and hormones to various organs in the human body, and plays an important role in the growth, development, and function maintenance of tissues and organs throughout the body.

The cardiovascular system exchanges nutrients and oxygen for various organs in the human body. In specific cardiovascular diseases, necrotic blood vessels sometimes need to be replaced and transplanted. However, the artificial blood vessels currently used as surgical transplants still cannot perfectly meet the requirements of high biocompatibility, good mechanical properties, and the ability to contract and relax. For example, artificial blood vessels currently used for coronary vascular transplantation with a diameter of less than 6 mm still suffer from low patency problems. At the same time, it is also difficult to customize and manufacture complex-shaped vascular structures at this stage. Therefore, blood vessel construction has always been a common problem in biofabrication of tissues and organs.

The main function of human bile ducts is to transport bile secreted by liver cells to the duodenum to help digest fatty foods. Bile duct obstruction causes cholestasis, which causes acute obstructive suppurative cholangitis, which can be life-threatening. At this time, the use of bioengineered bile ducts may serve as an alternative to liver transplantation. Therefore, the construction of bioengineered bile ducts with characteristics such as the structure, structural characteristics, markers and functions (alkaline phosphatase and γ-glutamyl transferase activity) of human bile ducts has also become a major demand.

As a tube connecting the larynx and bronchi, the human trachea is not only a passage for air, but also has the functions of defense, removal of foreign matter, and regulation of air temperature and humidity. Laryngotracheal stenosis or defect is a disabling disease that seriously affects people's quality of life. Resection of the diseased area is usually required, and the problem of healing after resection is a major clinical problem. Constructing a bioengineered trachea with tracheal structural characteristics and functions may be the key to solving this problem.

The human pancreatic duct drains pancreatic juice to the duodenum to help digest food. Blockage of the pancreatic duct leads to poor drainage of pancreatic juice, which can induce acute pancreatitis. In addition, the human body has other vascular organs such as ureters, lymphatic vessels, intestinal tubes, etc. Once these vascular organs become diseased, it may seriously affect human health. At present, the use of tubular biological structures for in vitro transplantation has become a common method. From this point of view, the construction of tubular tissues with certain mechanical properties and biological properties has become a major clinical demand and is also the focus of tissue engineering research.

Bio-3D printing is defined as the use of materials containing active cells for 3D printing technology. Bioprinting technology can arrange a large number of active cells and active biological materials in pre-designed spatial positions based on bionic principles and computer design, showing great advantages in the construction of various tissues and organs of the human body. However, printing tubular tissues with personalized structural dimensions (diameter, thickness, length, curvature, etc.) and corresponding mechanical and biological properties that meet clinical needs is still an urgent problem to be solved. Therefore, there is an urgent need to develop new cell printing processes for more complex in vitro tissues, especially longer tubular structures, such as blood vessels, trachea, bile ducts, larynxes, etc. Achieve separate control and controllable transportation of multiple materials, as well as stable manufacturing of longer tubular structures.

Contents of the invention

The object of the present invention is to provide a tubular tissue-like structure and a construction method thereof.

The concept of the invention is as follows: horizontal winding cell 3D printing technology is used to control the mixing and transportation of printing bio-ink, cross-linking agent, active cells and other substances. A slowly rotating rod-shaped collection device is used as the forming platform to construct a horizontal winding pattern of printing ink to form a long hollow tubular tissue-like structure.

In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a method for constructing a tubular tissue-like structure, which includes the following steps:

A. Prepare printing ink;

B. Culture the cells used for printing in vitro to obtain cell culture fluid;

C. Connect the printing ink and cell culture fluid to different nozzles of the bioprinter, and print them together on the winding rod to form a seamless tubular tissue. Then remove the winding rod, which is a hollow tubular tissue-like structure.

Among them, the material of the printing ink is a temperature-sensitive material and/or a biological material with good cytocompatibility and biocompatibility; the biological material uses one or more natural biological materials and/or synthetic biological materials.

The natural biological material is selected from the group consisting of gelatin, gelatin derivatives, alginates, alginate derivatives, cellulose, cellulose-derived materials, agar, Matrigel, collagen, collagen derivatives, amino acids, amino acid derivatives, proteoglycans , proteoglycan derivatives, glycoproteins and derivative materials, hyaluronic acid, hyaluronic acid derivatives, chitosan, chitosan derivatives, DNA hydrogel materials, laminin, fibronectin, fibrin, silk At least one of fibroin, silk fibroin derivatives, etc. Fibrin derivatives are preferred.

The artificial synthetic biological material is selected from polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid alkyd copolymer, Polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, poly At least one of ethylene, polycarbonate, polyethylene oxide, etc. Polylactic acid or lactic acid-glycolic acid copolymer is preferred.

In the aforementioned method, the cells described in step B can be vascular cells, and the vascular cells are selected from the group consisting of vascular endothelial cells, vascular endothelial progenitor cells, microvascular endothelial cells, vascular smooth muscle cells, vascular fibroblasts, mesenchymal stem cells, and pericytes. At least one of the others. Vascular endothelial cells and mesenchymal stem cells are preferred.

Wherein, the vascular cells are extracted from tissues or differentiated from stem cells.

The winding rod used in the aforementioned method may be a glass rod.

Preferably, the distance between the nozzle and the winding rod is 3-4mm.

The speed of the motor driver that drives the winding rod to rotate is 0-10000cts/s, and the speed of the motor driver that drives the printing nozzle to move horizontally is 0-10000cts/s.

In a second aspect, the present invention provides a tubular tissue-like structure constructed according to the above method.

The tubular tissue structure provided by the present invention can be used to construct a three-dimensional in vitro biological model of the tubular structure, conduct physiological and pathological analysis and research, and in vitro drug testing.

In a third aspect, the present invention provides a method for constructing a tubular blood vessel-like structure, including the following steps:

1) Dissolve 0.02g bovine blood fibrinogen (MACKLIN, F823833-1g) in 500 μl of DMEM/F-12 HEPES (MACKLIN, F6519-500ml) culture medium to obtain a fibrinogen solution;

2) Human umbilical vein endothelial cells HUVEC were cultured in vitro with EBM-2 Endothelial Growth Basal Medium (LONZA, CC-3156) and passaged. Before printing, culture to the fourth passage. Wash the cells with PBS, and then add Trypsin to the culture flask. -EDTA (Sigma, 59417C-500ML), digest in a 37°C incubator for 2 minutes, then add EBM (LONZA, CC-3156) culture medium to terminate digestion; put it into a centrifuge for centrifugation, discard the supernatant, and reuse the culture medium Suspend the cells, count and centrifuge again, discard the supernatant, and add the fibrinogen solution in step 1) to the cell pellet to obtain a cell-containing fibrinogen solution of 4×10 ⁶ cells/ml, which is used as printing reagent 1;

3) Use DMEM/F-12HEPES (MACKLIN, F6519-500ml) culture medium to prepare 20U/ml thrombin stock solution (bovine thrombin); before printing, add 0.12g anhydrous calcium chloride (can be replaced with glutaraldehyde, Add 500 μl of thrombin stock solution to carbodiimide or glycine), dissolve it and incubate it in a 37°C incubator for 5 minutes as printing reagent 2;

4) Connect printing reagents 1 and 2 to different nozzles of the bioprinter, and print them together on the winding rod to form a seamless tubular tissue. Then remove the winding rod to obtain a hollow tubular blood vessel-like structure.

Among them, the distance between the nozzle and the winding rod in step 4) is 3-4mm.

The speed of the electric driver that drives the winding rod to rotate is 0-10000cts/s (preferably 100cts/s, that is, one revolution every 100 seconds), and the speed of the motor driver that drives the printing nozzle in translation is 0-10000cts/s (preferably 80cts/s, that is, 125 seconds per revolution).

In a fourth aspect, the present invention provides a method for constructing a tubular bile duct-like structure, including the following steps:

1) Dissolve 0.02g bovine blood fibrinogen in 500 μl of DMEM/F-12HEPES (MACKLIN, F6519-500ml) culture medium to obtain a fibrinogen solution;

2) Preparation of printing reagent 1:

① hPSCs (human pluripotent stem cells) culture: Take an appropriate amount of hPSCs cells (Stemcell, 5795) for recovery and culture. The culture medium used is: activin A100ng/ml, bFGF 80ng/ml, BMP-4 10ng/ml, LY29400210μM and CHIR99021 3μM. , cultured overnight at 37°C;

② Differentiation culture of hPSCs into DE (definitive endoderm) cells: The next day, use activin A (Abcam, ab113316) 100ng/ml, bFGF (Beyotime, P6443-100μg) 80ng/ml, BMP-4 10 (Sigma, RAB0030) -1KT) ng/ml and LY294002 (CST, 9901S) 10 μM CDM–PVA culture medium to replace the culture medium in ① and culture at 37°C overnight; on the third day, use activin A (Abcam, ab113316) 100 ng/ml and bFGF added (Beyotime, P6443-100μg) 80ng/ml RPMI/B27 culture medium, replace the old culture medium;

③ Differentiation of DE cells into FP cells (foregut progenitor cells): On days 4-6, culture with RPMI (MACKLIN, R6516-500ml)/B27 (Sigma, SCM013) supplemented with activin A (Abcam, ab113316) 50ng/ml Replace the old culture medium with liquid; on days 7-8, replace the old culture medium with RPMI/B27 culture medium supplemented with activin A 50ng/ml;

④ Differentiation of FP cells into HB cells (hepatoblasts): On days 9-12, use RPMI/B27 containing SB-431542 (Sigma, S4317-5MG) 10 μM and BMP-4 (Sigma, RAB0030-1KT) 50ng/ml. The culture medium replaces the old culture medium; the differentiation of HB cells is detected by measuring the expression of HNF4A, AFP and TBX3 genes and flow cytometry analysis;

⑤ Differentiation of HB cells into CP (biliary epithelial progenitor cells): On days 13-16, use RPMI containing FGF10 (DLDEVELOP, DL-FGF10-Hu) 50ng/ml, activin A50ng/ml and retinoic acid (Beyotime, AF2398) 3μM /B27 culture medium replaces the old culture medium, and detects the differentiation of CP cells by measuring the expression of Sox9 gene;

⑥Rinse the CP cells with PBS, add cell digestion solution, incubate in a 37°C incubator for 20 minutes, collect the cells with a pipette; transfer the cells to RPMI/B27 culture medium, resuspend the cells, and then centrifuge at room temperature for 3 minutes, discard the supernatant, and resuspend the cells in 50% Matrigel matrix gel (BD, XYHZ-267) containing EGF (peprotech, AF-100-15-100) 20ng/ml and Rho kinase inhibitor Y-2763 210μm. After counting, centrifuge again, discard the supernatant, and add the fibrinogen solution in step 1) to the cell pellet to obtain a cell-containing fibrinogen solution of 4×10 ⁶ cells/ml, which is used as printing reagent 1;

3) Use DMEM/F-12HEPES culture medium to prepare 20U/ml thrombin stock solution (bovine thrombin); before printing, add 0.12g anhydrous calcium chloride (can be replaced with glutaraldehyde, carbodiimide or aminoacetic acid ), add 500 μl of thrombin stock solution, dissolve it and place it in a 37°C incubator for 5 minutes to serve as printing reagent 2;

4) Connect printing reagents 1 and 2 to different nozzles of the bioprinter, and print them together on the winding rod to form a seamless tubular tissue, and then remove the winding rod to obtain a hollow tubular three-dimensional structure;

5) Place the tubular three-dimensional structure of step 4) in WE (Sigma, W2895-1MG) culture medium containing 20ng/ml EGF. Change the medium every 2 days. After 2-4 days of culture, a tubular bile duct-like structure will be formed. .

Among them, the distance between the nozzle and the winding rod in step 4) is 3-4mm.

The speed of the electric driver that drives the winding rod to rotate is 0-10000cts/s (preferably 100cts/s, that is, one revolution every 100 seconds), and the speed of the motor driver that drives the printing nozzle in translation is 0-10000cts/s (preferably 80cts/s, that is, 125 seconds per revolution).

In the present invention, the fibrin hydrogel can be obtained through the reaction of fibrinogen and thrombin, and is an enzymatic cross-linked hydrogel. The specific mechanism is: after thrombin is mixed with fibrinogen, it will excise the two polypeptides at the ends of the α and β chains on fibrinogen to form fibrin monomers, and the fibrin monomers are autonomously cross-linked to form fibers due to the action of hydrogen bonds. Protein hydrogel.

In the present invention, other systems such as fibrin and gelatin mixtures, alginic acid and collagen mixtures, etc. can also be used to replace the above-mentioned fibrinogen-thrombin-calcium chloride system.

In a fifth aspect, the present invention provides a method for constructing a tubular bronchus-like structure, including the following steps:

1) Prepare printing ink

Gelatin (Sigma-Aldrich, G1890) and sodium alginate (Sigma-Aldrich, A0682) were respectively dissolved in 0.5% w/v sodium chloride solution to form a 15% gelatin solution and 4% sodium alginate. Solution; mix 600 μL of gelatin solution and 400 μL of sodium alginate solution, and incubate the resulting mixture at 37°C for 20 minutes to obtain printing ink;

2) Preparation of printed cells

Use H-DMEM medium (Hyclone, SH30022.01) containing 10% FBS to culture human lung bronchial epithelial cells and human fetal lung fibroblasts respectively; when the cells grow to cover 80% to 90% of the bottom of the dish, use H-DMEM medium (Hyclone, SH30022.01) containing 0.04 % EDTA and 0.25% trypsin (TargetMol, T0517-50mg) were used to digest the cells, passaged at a ratio of 1:6, and the culture medium was replaced every other day; cultured to the fourth generation before printing; then, human lung bronchial epithelial cells and Human fetal lung fibroblasts were mixed at a ratio of 5:1 to obtain a cell culture medium with a cell density of 6×10 ⁵ cells/ml;

3) Connect the printing ink of 1) and the cell culture solution of 2) to different nozzles of the bioprinter, and print them together on the winding rod to form a seamless tubular tissue. Then remove the winding rod to obtain a hollow tubular bronchus-like structure. body.

Among them, the distance between the nozzle and the winding rod in step 3) is 3-4mm.

The speed of the electric driver that drives the winding rod to rotate is 0-10000cts/s (preferably 100cts/s, that is, one revolution every 100 seconds), and the speed of the motor driver that drives the printing nozzle in translation is 0-10000cts/s (preferably 80cts/s, that is, 125 seconds per revolution).

The invention provides a tubular tissue-like structure and a construction method thereof. This method uses horizontal winding cell 3D printing technology to form a tubular structure with controllable structure and cell/material composition. The tubular structure is close to the shape and size of human blood vessels, has mechanical and biological properties that meet the needs of the human body, and has clinical application potential. The tubular tissue-like structure provided by the invention can be used in drug testing, tissue engineering, regenerative medicine, in vitro physiological model/pathological model/pharmacological model construction, cell structure construction, organoid construction, tissue/organ/human body chips, etc.

Through the above technical solutions, the present invention has at least the following advantages and beneficial effects:

(1) The core components of the present invention are the 3D printing nozzle and the winding rod. The printing nozzle design is flexible and the printing methods are diverse. It improves the utilization rate of printing materials and reduces the configuration requirements of printing materials. It expands the application range and combination possibilities of printable materials, so it has wide applicability and is conducive to the construction of printing materials with more complex structures and functions.

(2) The tubular tissue of the present invention can be used in replacement and bypass surgeries of various diseased parts of the vasculature of the human body, and is biocompatible.

(3) The tubular tissue of the present invention can be produced in batches. By improving the printing platform, the difficulty of constructing the tubular structure can be reduced, the construction time can be reduced, the construction cost of the tubular tissue can be reduced, and more patients can have the opportunity to receive treatment for tubular diseases. .

(4) The tubular tissue of the present invention can be constructed in a personalized manner. According to the needs, by adjusting the printing ink ratio and cell type, tubular tissue suitable for different types of vascular systems of the human body can be constructed.

(5) The cells used in the tubular tissue of the present invention can be autologous cells to construct a tubular structure that can adapt to the physiological environment of the human body without immune rejection, and is more suitable for repairing diseased parts of the human vasculature system.

(6) In the construction of tubular tissue of the present invention, individualized tubular tissue with structural dimensions (diameter, thickness, length, curvature, bifurcation, etc.) can be constructed by changing the operating mode of the printing nozzle and changing the shape and size of the winding rod. .

(7) The tubular tissue constructed by the present invention can be constructed by changing the ratio of printing materials to construct a tubular tissue with directional or staggered protein fiber arrangement, so that the tubular tissue has mechanical properties close to those of human tissue.

(8) The present invention uses a combination of the translation mode of the 3D printing nozzle and the rotation mode of the winding rod unit to construct a tubular structure. In this method, the module is simple to construct, easy to assemble, detachable, reusable, and has better operability.

(9) The present invention uses a wound rod unit to construct a tubular structure. In this method, the wound rod unit can be a tubular structure with certain mechanical properties and biological properties, so as to construct a composite with multi-layered structure, mechanical properties and biological properties. Tubular tissue.

Description of drawings

Figure 1 is a schematic flow chart of the cell printing method of the present invention.

Figure 2 shows the form of the printed cell-bearing structure soaked in the culture medium in Example 1 of the present invention.

Figure 3 is a Day 6 optical microscope picture of the tubular tissue-like structure constructed in Example 1 of the present invention.

Figure 4 is a CD31 staining diagram of the tubular tissue-like structure constructed in Example 1 of the present invention, and a schematic diagram of the Confocal layer scanning three-dimensional reconstruction.

Figure 5 is a statistical result of endothelial cell budding in printed structures in Example 1 of the present invention. Shown in the figure is the cell budding length per unit area at different times of culture.

Figure 6 is a CK19 immunostaining diagram of bile duct epithelial progenitor cells in the tubular bile duct-like structure in Example 2 of the present invention.

Detailed ways

The following examples are used to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

This invention utilizes the precise control of trace substances and cross-linking parameters by a 3D printing nozzle, as well as a horizontal rotating winding receiving device, to complete the cell 3D printing technology of instant printing and constructing a tubular structure.

As shown in Figure 1, the three-dimensional cell printing method of the present invention for constructing tubular structures includes the following steps:

1) Preparation of printing platform

The tubular structure constructed by the present invention mainly relies on the construction of a winding printing device. The design of the winding printing equipment is mainly divided into two aspects, the winding rod that can perform precise rotational movement and the printing nozzle that moves accurately. The translation direction of the printing nozzle is parallel to the winding rod and located in the same vertical direction. At the same time, the winding rod and the printing nozzle are controlled by an independent motor drive system to ensure that the speed of the two is independent and adjustable.

2) Preparation of printing ink (bioprinting material, printing matrix)

Prepare printing ink and set the ink distribution plan (such as composition, volume ratio, etc., but not limited to this). Prepare cells for printing. A power drive system (pneumatic method, but not limited to this) is used to push the printing nozzle to eject printing ink.

3) Printing parameter settings

Turn on the printing mode and control the printing process. According to the size (diameter, thickness, length) of the printed tubular structure, set the ejection speed of the printing ink in the 3D printing nozzle, set the running speed of the winding rod, and set the translation running speed of the printing nozzle. The printing ink in the 3D printing nozzle is pushed out through the nozzle and begins to adhere to the winding rod by gravity. At the same time, the wrapping rod rotates in real time, and the filamentous mixture extruded from the nozzle begins to wrap along the wrapping rod. At the same time, the translational movement of the printing nozzle and the rotational movement of the winding rod are combined to complete the printing of tubular structures of specific sizes.

4) Printed product inspection and parameter correction

The initially printed tubular structure is removed and its shape and structural size are detected. Compare the difference with the target tubular structure and correct the printing parameters. Repeat steps 1-4 to print again.

5)Cultivation of printed finished products

By controlling the printing time, a complete tubular structure of corresponding length is printed. The complete tubular tissue is placed in the culture medium for long-term dynamic culture.

6) Test the physical, chemical and biological properties of tubular tissues

After the tubular tissue is cultured in the culture medium for a period of time, it is taken out and the mechanical properties and biological properties are tested.

In a preferred embodiment, the printing platform in step 1) can adopt a variety of construction forms, such as frame type, ceiling type and other assembled construction forms, to form a printing platform with stable overall structure and convenient operation, so as to ensure The printing process is smooth and printing errors are reduced.

In a preferred embodiment, the 3D printing nozzle in step 1) can adopt various forms of nozzles, such as single-axis hollow nozzles, double hollow nozzles, and microfluidic chip nozzles based on microfluidic principles.

In a preferred embodiment, the printing nozzles in step 1) can be arranged in multiple numbers to complete printing of single-layer and multi-layer tubular structures.

In a preferred embodiment, the winding rod in step 1) can be changed to a clamping method with a certain inclination angle to the horizontal direction to print a tubular structure with a gradient thickness.

In a preferred embodiment, the winding rod in step 1) can be replaced with a luminal structure that has been previously cultured through tissue engineering and has a certain structural strength and biological activity to construct multi-level biological properties. A composite tubular tissue-like structure.

In a preferred embodiment, the winding rod in step 1) can be inserted into the lumen structure to construct a composite tubular tissue-like structure with a multi-layer lumen structure.

In a preferred embodiment, the combination of the movement of the printing nozzle and the winding rod in step 1) can be a translational movement of the printing nozzle and a fixed rotation of the winding rod. Or the printing nozzle is fixed and the winding rod rotates. Or both can travel simultaneously to complete the printing of tubular structures.

In a preferred embodiment, the printing nozzle in step 1) can be set to reciprocate to complete printing of single-layer and multi-layer tubular structures.

In a preferred embodiment, the rotational movement of the winding rod in step 1) can change the rotation direction of the winding rod, or set the rotation direction to alternately change over time to build a fiber with different fiber arrangement patterns or uneven thickness. Tubular tissue.

In a preferred embodiment, the rotation speed of the winding rod in step 1) can be adjusted by itself, and the translation speed of the printing nozzle can be adjusted by itself. The two speeds can be combined independently to achieve the printing of tubular structures of different structures and sizes.

In a preferred embodiment, the rotation speed of the winding rod and the translation speed of the printing nozzle in step 1) are self-adjustable. The adjustment method can be achieved by controlling the working status of the drive motor or adding a speed conversion joint.

In a preferred embodiment, in step 1), the printing nozzle can use three-dimensional drawing software (such as Solidworks, but not limited to this) to design the three-dimensional structural diagram of the 3D printing nozzle; use existing materials (such as PDMS, PMMA, but not limited thereto) processing (such as molding, but not limited thereto) processing and shaping, but not limited thereto.

In a preferred embodiment, the winding rod in step 1) can be made of one or more materials such as glass, resin, etc. Its cross-sectional configuration may be one or more of circular, elliptical, or polygonal. Its diameter can be a single diameter or vary with the axis direction. The center line can be a straight line or a curve. Through different settings of the winding rod, different configurations of tubular structures can be printed.

In a preferred embodiment, the printing ink material in step 2) is a mixture of temperature-sensitive materials and/or other biological materials with good cytocompatibility and biocompatibility; wherein, the biological material can be a mixture of One or more natural biological materials and/or synthetic biological materials.

In some embodiments, the natural biological material used in the printing ink in step 2) is at least one of the following materials: gelatin, gelatin derivatives, alginate (such as sodium alginate), alginate derivatives materials, cellulose, cellulose-derived materials, agar, Matrigel, collagen, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derivative materials, hyaluronic acid, hyaluronic acid derivatives , chitosan, chitosan derivatives, DNA hydrogel materials, laminin, fibronectin, fibrin, silk fibroin, silk fibroin derivatives, more preferably fibrin derivatives.

In some embodiments, the synthetic biological material used in the printing ink in step 2) is at least one of the following materials: polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, Polylactic acid, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid alkyd copolymer, polydimethylsiloxane, polyanhydride, polyester, polyamide, polyamino acid, polyacetal, polycyano Acrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, preferably polylactic acid or lactic acid-glycolic acid copolymer.

In some embodiments, the vascular cells used in the printing ink in step 2) include vascular endothelial cells, vascular endothelial progenitor cells, microvascular endothelial cells, vascular smooth muscle cells, vascular fibroblasts, mesenchymal stem cells, and pericytic cells. Cells, these cells can be extracted from tissues or differentiated from stem cells, preferably vascular endothelial cells and mesenchymal stem cells.

In a preferred embodiment, in step 3), the printing ink can complete the reaction (cross-linking reaction, but not limited to this) before, during and after the printing nozzle pushing action to form an available Wrapped filamentous polymer to complete a tubular tissue with a fibrous structure.

In some embodiments, the physical and chemical properties and biological properties in step 6) can be measured using (uniaxial tensile test, immunofluorescence test, but not limited to) and other experimental methods.

In a preferred embodiment (Embodiment 1), as shown in Figure 2, the medium used for tubular tissue culture is a medium that maintains one or more tissue configurations fixed, stable and structurally or functionally enhanced.

In a preferred embodiment (Embodiment 1), as shown in Figure 4, for detecting the morphology and size structure of tubular tissue, detection methods (optical microscope, scanning electron microscope, but not limited to this) can be applied.

Example 1 Construction of fibrin tubular blood vessel-like structure

1. Printing platform preparation

Construct a stainless steel alloy frame structure, assemble the 3D printing nozzle and the winding rod within the frame, debug the motor control system, and build a motion system to ensure that the 3D printing nozzle can perform translational motion and the winding rod can perform rotational motion. During the installation process, ensure that the 3D printing nozzle is placed directly above the winding rod and in the same vertical plane as the winding rod. The winding rod is placed horizontally, and its rotational movement can be accurately controlled. The translation direction of the 3D printing nozzle is along the axis of the winding rod, and its movement can also be accurately controlled. The platform structural parts are all customized from the company. A few parts, such as motor mounting plates, are obtained by purchasing raw materials such as plates, drawing and designing by ourselves, and entrusting professional machining units to process them.

This embodiment uses a microfluidic chip nozzle based on the microfluidic principle, and uses Solidworks structural design software to design the microfluidic chip structure diagram. The microfluidic flow channel configuration adopts the Y+S type. On the one hand, it is to increase the distance traveled by the mixed liquid and leave more room for changes in the pushing speed. On the other hand, the S-shaped channel is also conducive to the uniformity of the mixed liquid. mix. The convergence inlet of the mixing channel is rounded to prevent bubbles from being generated at sharp corners when the liquid flows. In order to avoid affecting the printing performance of the material, hydrophilic materials or coatings are selected for the nozzle material or channel surface to reduce the adsorption of fibrin gel and avoid channel blockage.

In this embodiment, a detachable PMMA chip printing nozzle is designed. A layer of pressure-sensitive adhesive is sandwiched between two pieces of PMMA and fixed with screws. At the same time, plastic joints are used, glue-sealed steel needles are used as guides, and PVC pipes are used as injection pipes, so as to have a smoother surface and effectively avoid liquid seepage.

The winding rod used in the winding rotation movement is a glass rod with a certain length and diameter.

2. Preparation of printing ink (bioprinting material)

The printing ink of the present invention can be purchased through commercial channels, or can be prepared according to actual needs. In this embodiment, the fibrin tubular blood vessel-like structure is printed, and the printing material (printing ink) is prepared before printing the fibrin tubular blood vessel-like structure. Specifically, The preparation process is:

1) Prepare the main material printing ink (bovine blood fibrinogen and thrombin)

Bovine blood fibrinogen needs to be stored in the refrigerator at -20°C for a long time. It should be prepared immediately when using it. When preparing bovine blood fibrinogen, the weighing balance should be sprayed with alcohol before being moved to a clean bench and sterilized by ultraviolet light for 30 seconds. Minutes, each printing mainly uses 0.02g of powder. Each time, take out a tube containing 0.02g of fibrinogen and dissolve it in 500μl of DMEM/F-12HEPES culture medium to ensure that the DMEM in the culture medium is completely dissolved and there is no precipitation.

Thrombin is first divided into several EP tubes with PBS. Each tube contains 100U in 1ml and is stored at -20°C. To prepare a batch of materials, you only need to take out one tube and use DMEM/F-12HEPES culture medium to dilute it to 20U/ ml of total 5ml of mother liquor, wrapped in tin foil to avoid light and stored in a 4°C refrigerator. One batch of mother liquor is enough to print multiple times. Each time you print, first take a new EP tube and add an anhydrous calcium chloride particle with a mass of about 0.12g, and add 500μl of thrombin stock solution and pipet until it is completely dissolved. After the configuration is completed, put the liquid into a 37°C incubator. 5 minutes.

The final concentrations used in this example are: fibrinogen: 20mg/ml, thrombin: 10U/ml, calcium chloride: 120mM. Use a 1ml needle to inhale the material before printing. Pay attention to the need to minimize the inhaled bubbles. After inhalation, the syringe can be tapped to eliminate air bubbles.

2) Preparation of cells in printing ink

The cells used for printing in this example are human umbilical vein endothelial cells (HUVEC), which are cultured using EBM-2 EndothelialGrowth Basal Medium (Lonza), and the passage number before printing is the fourth generation. Before printing, the T75 culture flask is rinsed with PBS, and Use 3 ml of 0.25% Trypsin-EDTA (Thermal Fisher) for 2 minutes in a 37°C incubator. After observing under the microscope, add 6 ml of EBM culture medium to terminate the digestion. Put it into a centrifuge for centrifugation. The parameters used are 1000 rpm. and process for 3 minutes, then remove the supernatant and add 1 ml of culture medium to resuspend the cells. Use a cell counting board to count. The cell concentration can be obtained and the total cell number can be estimated. After counting, take out the required amount of solution and centrifuge again. , the parameters are the same as the previous step, remove the supernatant and add 500 μl of the previously prepared fibrinogen solution to prepare a cell-containing fibrinogen solution with a concentration of 4×10 ⁶ cells/ml.

3. Print settings

All infusion work during the printing process is mainly completed using the Baoding Shenchen SPLab02 syringe pump. Before each printing, the flow channel is perfused with deionized water, and a total of 50 μl is rinsed at a flow rate of 5 μl/min. On the one hand, it is used as a pre-use flow. On the one hand, channel flushing is also to fill the flow channel with liquid, which can reduce resistance when adding materials and make the flow channel reach a steady state as soon as possible. After rinsing, connect the chip to the tubes for fibrinogen and thrombin respectively and pass liquid through them. The same setting is 5μl/min. The total volume is generally set to 400μl. You can use the fast forward button on the syringe pump in small amounts to make the flow channel Quickly reach a steady state, and when you see pink bead-like gel appearing under the chip, you can put the microfluidic chip nozzle on the printer beam. The distance between the nozzle and the winding glass tube is about 3-4mm.

In the printer parameter section, adjust the rotation speed of the winding rod to 100cts/s, that is, one revolution every 100 seconds, and the motor driver that drives the printing nozzle to move in translation, at a speed of 80cts/s, that is, one revolution every 125 seconds, to achieve stable printing without gaps. tubular tissue. A single printing can print a tubular blood vessel-like structure with a total length of 6cm and a wall thickness of 2mm.

4. Printed finished product inspection and parameter correction

Use an optical microscope and an electron scanning microscope to observe and detect the printed tubular structure, correct the printing parameters (rotation rate of the winding rod, ejection rate and translation speed of the microfluidic chip nozzle, etc.), and print again.

5. Print finished product cultivation

Place the printed fibrin hollow structure in a culture dish rich in culture fluid.

6. Perform vascularization testing on the physical, chemical and biological properties of tubular tissues

After the fibrin hollow structure is cultured in the culture medium for a period of time, it is taken out and the mechanical properties and biological properties are tested. Uniaxial tensile testing was used to measure the mechanical properties of fibrin hollow structures and compare them with the characteristics of human blood vessels. CCK8 cell proliferation was used to detect cell survival status, and immunofluorescence test (CD31 and DAPI) was used to detect cell proliferation and growth status.

Figure 3 is a Day 6 optical microscope picture of the tubular tissue-like structure constructed in this embodiment. Figure 4 shows that endothelial cells can spread and grow well in the printed oriented fibrin and form a vein-like structure. Figure 5 is a statistical result of endothelial cell budding in the printed structure. The results show that as the culture time increases, the length of endothelial cell budding increases, indicating that vascularization begins inside the tubular structure.

Example 2 Construction of fibrin tubular bile duct-like structure

1. Printing platform preparation

A stainless steel alloy frame structure printing platform is constructed, which mainly includes a printing nozzle and a winding unit for tubular construction. Among them, the printing platform and its components are independently drawn and designed, and each component can be customized by the enterprise or entrusted to a professional machining unit for processing.

In this embodiment, a microfluidic chip nozzle designed based on the microfluidic principle is used, and PMMA material is used to construct a microfluidic chip nozzle with a Y+S configuration flow channel. The nozzle can effectively promote solution fusion and prevent channel clogging problems.

The diameter of the winding glass tube used in this embodiment is about 0.5 cm, which is similar to the diameter of the human bile duct.

2. Preparation of printing ink (bioprinting material)

The printing ink of the present invention can be purchased through commercial channels, or can be prepared according to actual needs. In this embodiment, the fibrin tubular bile duct-like structure is printed, and the printing material (printing ink) is prepared before printing. The specific preparation process is:

1) Prepare the main material printing ink (bovine blood fibrinogen and thrombin)

Take 0.02g powder of bovine blood fibrinogen stored in the refrigerator at -20°C and dissolve it in 500 μl of DMEM/F-12HEPES culture medium to ensure that the DMEM in the culture medium is completely dissolved and there is no precipitation.

Thrombin is first divided into several EP tubes with PBS. Each tube contains 100U in 1ml and is stored at -20°C. To prepare a batch of materials, you only need to take out one tube and dilute it to 20U with DMEM/F-12 HEPES culture medium. /ml of a total of 5ml of mother liquor, wrapped in tin foil to avoid light and stored in a 4°C refrigerator. One batch of mother liquor is enough to print multiple times. Each time you print, first take a new EP tube and add an anhydrous calcium chloride particle with a mass of about 0.12g, and add 500μl of thrombin stock solution and pipet until it is completely dissolved. After the configuration is completed, put the liquid into a 37°C incubator. 5 minutes.

The final concentrations used in this example are: fibrinogen: 20mg/ml, thrombin: 10U/ml, calcium chloride: 120mM.

2) Preparation of cells in printing ink

The cells used for printing in this example are human bile duct epithelial progenitor cells (CP, cholangiocyte progenitor).

① hPSCs cell culture: Take an appropriate amount of hPSCs cells for recovery and culture for selection. On the first day, culture medium rich in ingredients activin A (100ng/ml), bFGF (80ng/ml), BMP-4 (10ng/ml), LY294002 (10μM) and CHIR99021 (3μM) was used to treat multifunctional hepatocyte hPSCs. Perform a medium change operation and incubate overnight at 37°C.

② Differentiation culture of hPSCs into DE: On the next day, replace the culture medium with CDM–PVA supplemented with activin A (100ng/ml), bFGF (80ng/ml), BMP-4 (10ng/ml) and LY294002 (10μM). Incubate overnight at 37°C. On the third day, use the new RPMI/B27 culture medium containing activin A (100ng/ml) and bFGF (80ng/ml) to replace the culture medium.

③ Differentiation of DE into FP cells: On days 4-6, replace the old culture medium with newly prepared RPMI/B27 culture medium supplemented with activin A (50ng/ml). On days 7 and 8, replace the culture medium with newly prepared RPMI/B27 culture medium supplemented with activin A (50ng/ml).

④ Differentiation of FP cells into HB cells: On days 9-12, replace the culture medium with newly prepared RPMI/B27 culture medium containing SB-431542 (10 μM) and BMP-4 (50 ng/ml). The differentiation of HB hepatic progenitor cells was detected through the expression of HNF4A, AFP and TBX3 and flow cytometry analysis. Realize the differentiation of FP cells into HB cells.

⑤ Differentiation of HBs cells into CPs: On days 13-16, replace the culture medium with newly prepared RPMI/B27 medium containing FGF10 (50ng/ml), activinA (50ng/ml) and retinoic acid (3μM). Expression to detect differentiation of bile duct epithelial progenitor cells. Ensure differentiation of hepatic progenitor cells into bile duct epithelial progenitor cells.

⑥Rinse the cells with PBS, add cell digestion solution, and store at 37°C for 20 minutes. The cells are detached from the bottom plate and collected using a pipette. Transfer the cells to a 15 ml tube, gently blow and resuspend the cells 2-3 times, and use a 1000 µl pipette to separate the cells into small clumps. Rinse plate with RPMI/B27 medium and transfer to 15 ml tube. Centrifuge at room temperature for 3 minutes. Aspirate the supernatant and resuspend the cells in 6 ml of RPMI/B27. Centrifuge for 3 minutes at room temperature and absorb the supernatant. Resuspend cells in previously prepared 50% matrix gel containing EGF (20ng/ml) and Rho kinase inhibitor Y-27632 (10μm) and mix thoroughly.

Use a cell counting board to count to obtain the cell concentration and estimate the total cell number. After counting, take out the required amount of cell solution and centrifuge again. The parameters are the same as in the previous step. Remove the supernatant and add 500 μl of fiber prepared previously. The proteinogen solution is prepared into a cell-containing fibrinogen solution with a concentration of 4×10 ⁶ cells/ml.

3. Print settings

Baoding Shenchen SPLab02 syringe pump is used to inject and pressure drive the printing ink. Before each printing, the flow channel is perfused with deionized water, so that the resistance can be reduced when adding materials and the flow channel can reach a steady state as soon as possible. After rinsing, connect the chip to the tubes of fibrinogen and thrombin respectively and pass liquid through them. Also set it to 5μl/min. The total volume is generally set to 400μl. Adjust the syringe pump to quickly reach a steady state in the flow channel. When you see pink bead-like gel appearing under the chip, you can put the microfluidic chip nozzle on the printer beam. The distance between the nozzle and the winding glass tube is about 3-4mm.

Adjust the printer parameters to ensure that tubular tissue without gaps can be stably printed.

4. Printed finished product inspection and parameter correction

Use an optical microscope and an electron scanning microscope to observe and detect the printed tubular structure, correct the printing parameters (rotation rate of the winding rod, ejection rate and translation speed of the microfluidic chip nozzle, etc.), and print again.

5. Print finished product cultivation

The printed fibrin bile duct structure was placed in freshly prepared EGF (20ng/ml) WE medium, and the medium was replaced every 2 days. Within 2-4 days of culture, organoid tissue will form.

6. Detect bile duct-like properties on the physical, chemical and biological properties of tubular tissue

After the fibrin hollow structure is cultured in the culture medium for a period of time, it is taken out and the mechanical properties and biological properties are tested. Differentiation of bile duct epithelial-like cells was detected by expression of CK7, ensuring that it could be observed in >75% of cells. The expression of CK19 was detected by immunofluorescence staining to characterize the growth of bile duct epithelial cells. Detection of alkaline phosphatase staining and glutyl transpeptidase activity used to characterize bile duct epithelial cell function. The CK19 immunofluorescence staining picture (Figure 6) shows the positive expression of CK19, indicating that the bile duct epithelial cells are growing well and have tubular structures.

Example 3 Construction of tubular bronchial-like structure

1. Printing platform preparation

A stainless steel alloy frame structure printing platform is constructed, which mainly includes a printing nozzle and a winding unit for tubular construction. Among them, the printing platform and its components are independently drawn and designed, and each component can be customized by the enterprise or entrusted to a professional machining unit for processing.

This embodiment uses a microfluidic chip nozzle designed based on the microfluidic principle, and uses PMMA material to construct a microfluidic chip nozzle with a single flow channel. The nozzle enables precise control of solution flow.

The diameter of the winding glass tube used in this embodiment is about 1 cm, which is similar to the diameter of human bronchus.

2. Preparation of printing ink (bioprinting material)

The printing ink of the present invention can be purchased through commercial channels, or can be prepared according to actual needs. In this embodiment, a tubular bronchus-like structure is to be printed, and the printing material (printing ink) is prepared before printing. The specific preparation process is:

1) Prepare the main material printing ink (alginic acid and gelatin)

Gelatin (Sigma-Aldrich, G1890) and sodium alginate (Sigma-Aldrich, A0682) were dissolved in 0.5% (w/v) sodium chloride solution to form a 15% gelatin solution and 4% alginic acid respectively. Sodium solution.

2) Preparation of cells in printing ink

The cells used in this example are human lung bronchial epithelial cells (Beas-2B) and human fetal lung fibroblasts (MRC-5).

Cell culture: Use H-DMEM medium (Hyclone, SH30022.01) (containing 10% FBS) for culture. When the cells have grown to cover about 80% of the bottom of the dish, digest them with 0.25% trypsin (TargetMol, T0517-50mg) (containing 0.04% EDTA), passage them at a ratio of 1:6, and replace the culture medium every other day.

3. Print settings

Incubate 600 μL of gelatin solution and 400 μL of sodium alginate solution at 37°C for 20 minutes and mix gently to serve as matrix material. Beas-2B and MRC-5 cells (cell density is 6×10 ⁵ cells/ml, the ratio of the two is 5:1).

Baoding Shenchen SPLab02 syringe pump is used to inject and pressure drive the printing ink. Before each printing, the flow channel is perfused with deionized water, so that the resistance can be reduced when adding materials and the flow channel can reach a steady state as soon as possible. After rinsing is completed, connect the chip to the above-mentioned mixed solution and pass the liquid through it. Also set it to 5 μl/min. The total volume is generally set to 400 μl. Adjust the syringe pump to quickly reach a steady state in the flow channel. The distance between the nozzle and the winding glass tube is about 3-4mm.

Adjust the printer parameters to ensure that tubular tissue without gaps can be stably printed.

4. Printed finished product inspection and parameter correction

Use an optical microscope and an electron scanning microscope to observe and detect the printed tubular structure, correct the printing parameters (rotation rate of the winding rod, ejection rate and translation speed of the microfluidic chip nozzle, etc.), and print again.

5. Print finished product cultivation

The printed bronchial-like structure was placed in a 5% CO ₂ , 37°C incubator and cultured in freshly prepared H-DMEM medium (containing 10% FBS), and the medium was replaced every 1-2 days.

6. Detect bronchiectasis on the physical, chemical and biological properties of tubular tissue

After the bronchus-like hollow structure is cultured in the culture medium for a period of time, it is taken out and the mechanical properties and biological properties are tested. After 7 days of growth after the construction of the bronchus-like structure, HE staining showed the phenomenon of cell connection growth; broad-spectrum CK and Vimentin staining both had positive results, indicating that the cell activity was good.

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims (3)

1. A method for constructing a tubular blood vessel-like structure, which is characterized by comprising the following steps: 1) Dissolve 0.02g bovine blood fibrinogen in 500μl DMEM/F-12HEPES culture medium to obtain a fibrinogen solution; 2) Human umbilical vein endothelial cells HUVEC were cultured in vitro and passaged using EBM-2Endothelial Growth Basal Medium. Culture them to the fourth passage before printing. Wash the cells with PBS, then add Trypsin-EDTA to the culture bottle and incubate at 37°C. Digest for 2 minutes, then add EBM culture medium to terminate digestion; put it into a centrifuge for centrifugation, discard the supernatant, resuspend the cells in culture medium, count and centrifuge again, discard the supernatant, and add the fiber from step 1) to the cell pellet. Proteinogen solution to obtain a cell-containing fibrinogen solution of 4×10 ⁶ cells/ml, which is used as printing reagent 1; 3) Use DMEM/F-12HEPES culture medium to prepare a 20U/ml thrombin stock solution; before printing, add 500 μl of the thrombin stock solution to 0.12g anhydrous calcium chloride, dissolve it and place it in a 37°C incubator for 5 minutes. As printing reagent 2; 4) Connect printing reagents 1 and 2 to different nozzles of the bioprinter, and print them together on the winding rod to form a seamless tubular tissue. Then remove the winding rod to obtain a hollow tubular blood vessel-like structure. 2. The method according to claim 1, characterized in that the distance between the nozzle and the winding rod in step 4) is 3-4mm; and/or The speed of the motor driver that drives the winding rod to rotate is 0-10000cts/s, and the speed of the motor driver that drives the printing nozzle to move horizontally is 0-10000cts/s. 3. The tubular blood vessel-like structure constructed according to the method of claim 1 or 2.