CN112206074A - Tubular tissue-like structure and method for constructing same - Google Patents
Tubular tissue-like structure and method for constructing same Download PDFInfo
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- CN112206074A CN112206074A CN201910549415.7A CN201910549415A CN112206074A CN 112206074 A CN112206074 A CN 112206074A CN 201910549415 A CN201910549415 A CN 201910549415A CN 112206074 A CN112206074 A CN 112206074A
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- A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
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Abstract
The invention provides a tubular tissue 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. the printing ink and the cell culture solution are respectively connected with different nozzles of a biological printer and are jointly printed on a winding rod to form a seamless tubular tissue, and then the winding rod is removed, so that the hollow tubular tissue structure is obtained. The method forms a tubular structure with controllable structure and cell/material composition by a horizontal wound cell 3D printing technique. The tubular structure is similar to the shape and the size of a human body vessel, has mechanical and biological properties suitable for human body requirements, has clinical application potential, and can be used for the aspects of drug detection, tissue engineering, regenerative medicine, in-vitro physiological model/pathological model/pharmacological model construction, tissue/organ/human body chips and the like.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to a tubular tissue-like structure and a construction method thereof.
Background
The human vascular system is widely distributed, provides transport of nutrient substances, oxygen, hormone and the like of various organs in the human body, and plays an important role in growth, development and function maintenance of tissues and organs of the whole body.
The cardiovascular system exchanges nutrients and oxygen with various organs in the human body. In specific cardiovascular diseases, the replacement transplantation of necrotic blood vessels is sometimes required, but the current artificial blood vessels used as surgical transplantation still cannot perfectly meet the requirements of high biocompatibility, good mechanical property, capability of contraction and relaxation and the like. For example, the current artificial blood vessels with diameter less than 6mm for coronary transplantation still have the problem of low patency rate, and at the same time, the current vascular structures with complex shapes are difficult to customize and manufacture. Thus, vascular architecture has also been a common problem in the biological manufacture of tissues and organs.
The bile duct of human body mainly acts to transport bile secreted by liver cells to duodenum, and helps to digest fatty food. Bile siltation caused by bile duct obstruction, acute obstructive suppurative cholangitis caused by bile duct obstruction, and life threatening may occur. At this time, the use of the bioengineered bile duct may be an alternative to liver transplantation. Therefore, it is also a great demand to construct bioengineering bile ducts with the characteristics of human bile duct structure, structural characteristics, markers and functions (alkaline phosphatase and gamma-glutamyltransferase activity).
The human trachea is used as a pipeline connecting the larynx and the bronchus, is not only a passage of air, but also has the functions of defending, removing foreign matters and adjusting the temperature and the humidity of the air. Laryngotracheal stenosis or defects is a disabling disease that seriously affects the quality of life of people. Resection of the lesion is often required, and post-resection healing is a major clinical problem. The key to solve the problem is to construct a bioengineering trachea with the structural characteristics and functions of the trachea.
The human pancreatic duct drains pancreatic juice to the duodenum, helping to digest food. Pancreatic duct blockage can cause poor drainage of pancreatic juice, which can induce acute pancreatitis. In addition, the human body has other tubular organs such as ureter, lymphatic vessel, intestinal vessel, etc. once these vascular organs are diseased, the health of the human body may be seriously affected. At present, the in vitro transplantation by using the tubular biological structure body is a common method. Therefore, the construction of a tubular tissue with certain mechanical properties and biological properties is a great clinical requirement, and is also a research focus of tissue engineering.
Biological 3D printing, defined as the use of material containing viable cells for 3D printing technology. The biological printing technology can arrange a large amount of active cells and active biological materials at a pre-designed spatial position according to a bionic principle and computer design, and has great advantages in the construction of various tissues and organs of a human body. However, it is still a problem to be solved that the printing of the tubular tissue with personalized structure size (diameter, thickness, length, curvature, etc.) and corresponding mechanical and biological properties, etc. to meet clinical requirements is still needed. Therefore, development of new cell printing processes is urgently needed for more complex in vitro tissues, particularly for longer tubular structures, such as blood vessels, trachea, bile ducts, laryngeal tubes, and the like. Realizing the respective control and controllable delivery of various materials and the stable manufacture of a longer tubular structure.
Disclosure of Invention
The invention aims to provide a tubular tissue structure and a construction method thereof.
The invention has the following conception: and the mixing and conveying of printing biological ink, a cross-linking agent, active cells and other substances are controlled by adopting a horizontal winding type cell 3D printing technology. A slowly rotating rod-shaped collecting device is used as a forming platform, a printing ink horizontal winding mode is constructed, and a hollow tubular tissue structure body with a long length is formed.
In order to achieve the object of the present invention, in a first aspect, the present invention provides a method for constructing a tubular tissue-like structure, comprising the steps of:
A. preparing printing ink;
B. culturing cells for printing in vitro to obtain a cell culture solution;
C. the printing ink and the cell culture solution are respectively connected with different nozzles of a biological printer and are jointly printed on a winding rod to form a seamless tubular tissue, and then the winding rod is removed, so that the hollow tubular tissue structure is obtained.
Wherein, the printing ink is made of temperature-sensitive material and/or biological material with good cell compatibility and biocompatibility; the biological material is one or more natural biological materials and/or artificially synthesized biological materials.
The natural biomaterial is at least one selected from gelatin, gelatin derivatives, alginate derivatives, cellulose-derived materials, agar, matrigel, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derived materials, hyaluronic acid derivatives, chitosan derivatives, DNA hydrogel materials, layer-linked proteins, fibronectin, fibrin, silk fibroin derivatives, and the like. Fibrin derivatives are preferred.
The artificial biological material is at least one selected from polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide and the like. Polylactic acid or lactic acid-glycolic acid copolymer is preferred.
In the aforementioned method, the cell in step B may be a vascular cell selected from at least one of vascular endothelial cell, vascular endothelial progenitor cell, microvascular endothelial cell, vascular smooth muscle cell, vascular fibroblast, mesenchymal stem cell, pericyte and the like. Vascular endothelial cells and mesenchymal stem cells are preferred.
Wherein the vascular cells are obtained by extraction from a tissue or are differentiated from stem cells.
The wrapping rod used in the aforementioned method may be a glass rod.
Preferably, the distance between the spray head and the winding rod is 3-4 mm.
The rotating speed of the motor driver for driving the winding rod to rotate is 0-10000cts/s, and the rotating speed of the motor driver for driving the printing nozzle to translate is 0-10000 cts/s.
In a second aspect, the present invention provides a tubular tissue-like structure constructed according to the above method.
The tubular tissue-like structure provided by the invention can be used for constructing a three-dimensional in vitro biological model of a tubular structure, analyzing and researching physiology and pathology and testing in vitro drugs.
In a third aspect, the present invention provides a method for constructing a tubular vascular structure, comprising the steps of:
1) dissolving 0.02g bovine fibrinogen (MACKLIN, F823833-1g) in 500. mu.l DMEM/F-12HEPES (MACKLIN, F6519-500ml) culture solution to obtain fibrinogen solution;
2) HUVEC (human umbilical vein Endothelial cells) are cultured in vitro by using EBM-2Endothelial Growth basic Medium (LONZA, CC-3156) and passaged, the cells are washed by PBS (PBS) before being cultured to the fourth generation, then Trypsin-EDTA (Sigma, 59417C-500ML) is added into a culture bottle, the cells are digested for 2 minutes in an incubator at 37 ℃, and then EBM (LONZA, CC-3156) culture solution is added to stop digestion; centrifuging in a centrifuge, removing supernatant, resuspending cells with culture medium, counting, centrifuging again, removing supernatant, adding fibrinogen solution obtained in step 1) into cell precipitate to obtain 4 × 106cells/ml of fibrinogen solution containing cells as printing reagent 1;
3) preparing 20U/ml thrombin mother liquor (bovine thrombin) by using DMEM/F-12HEPES (MACKLIN, F6519-500ml) culture solution; before printing, adding 500 mu l of thrombin mother liquor into 0.12g of anhydrous calcium chloride (which can be replaced by glutaraldehyde, carbodiimide or glycine), dissolving, and then putting into an incubator at 37 ℃ for incubation for 5 minutes to serve as a printing reagent 2;
4) and respectively connecting the printing reagents 1 and 2 with different nozzles of a biological printer, printing the printing reagents on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain the hollow tubular vascular-like structure.
Wherein, the distance between the spray head and the winding rod in the step 4) is 3-4 mm.
The motor driver for driving the winding rod to rotate has a rotation speed of 0-10000cts/s (preferably 100cts/s, i.e. one rotation in 100 seconds), and the motor driver for driving the printing nozzle to translate has a rotation speed of 0-10000cts/s (preferably 80cts/s, i.e. one rotation in 125 seconds).
In a fourth aspect, the present invention provides a method for constructing a tubular bile duct-like structure, comprising the steps of:
1) dissolving 0.02g bovine fibrinogen in 500 μ l DMEM/F-12HEPES (MACKLIN, F6519-500ml) culture solution to obtain fibrinogen solution;
2) preparation of printing agent 1:
culturing hPSCs (human pluripotent stem cells): an appropriate amount of hPSCs (Stemcell, 5795) were used for recovery culture in the following culture medium: 100ng/ml activin A, 80ng/ml bFGF, 410 ng/ml BMP-410 ng/ml LY 29400210. mu.M and CHIR 990213. mu.M, and culturing overnight at 37 ℃;
② the differentiation culture of hPSCs to DE (definitive endoderm) cells: the next day, the culture solution of the first is replaced with CDM-PVA culture solution added with activin A (Abcam, ab113316)100ng/ml, bFGF (Beyotime, P6443-100 μ g)80ng/ml, BMP-410 (Sigma, RAB0030-1KT) ng/ml and LY294002(CST, 9901S)10 μ M, and cultured overnight at 37 ℃; on the third day, the culture medium was replaced with RPMI/B27 supplemented with activin A (Abcam, ab113316) at 100ng/ml and bFGF (Beyotime, P6443-100. mu.g) at 80 ng/ml;
③ differentiation of DE cells into FP cells (foregut progenitors): on days 4-6, the old culture medium was replaced with RPMI (MACKLIN, R6516-500ml)/B27(Sigma, SCM013) supplemented with activin A (Abcam, ab113316)50 ng/ml; on days 7-8, replacing the old culture medium with RPMI/B27 supplemented with activin A50 ng/ml;
(iv) differentiation of FP cells into HB cells (hepatoblasts): on days 9-12, the old culture medium was replaced with RPMI/B27 containing 10. mu.M of SB-431542(Sigma, S4317-5MG) and 50ng/ml of BMP-4(Sigma, RAB0030-1 KT); detecting the differentiation of HB cells by measuring the expression of HNF4A, AFP and TBX3 genes and flow analysis;
differentiation of HB cells into CP (biliary epithelial progenitor cells): on days 13-16, the old culture medium was replaced with 3. mu.M RPMI/B27 containing FGF10(DLDEVELOP, DL-FGF10-Hu)50ng/ml, activin A50 ng/ml and retinoic acid (Beyotime, AF2398), and the differentiation of CP cells was examined by measuring the expression of Sox9 gene;
sixthly, washing CP cells with PBS, adding cell digestive juice, and culturing at 37 deg.CIncubating for 20 minutes in the incubator, and collecting cells by using a pipette; transferring the cells to RPMI/B27 culture solution, resuspending the cells, centrifuging at room temperature for 3 min, discarding the supernatant, resuspending the cells in 50% Matrigel (BD, XYHZ-267) containing EGF (peprotech, AF-100-15-100)20ng/ml and Rho kinase inhibitor Y-2763210 μm, counting, centrifuging again, discarding the supernatant, adding the fibrinogen solution of step 1) to the cell pellet to obtain 4X 106cells/ml of fibrinogen solution containing cells as printing reagent 1;
3) preparing 20U/ml thrombin mother liquor (bovine thrombin) by using DMEM/F-12HEPES culture solution; before printing, adding 500 mu l of thrombin mother liquor into 0.12g of anhydrous calcium chloride (which can be replaced by glutaraldehyde, carbodiimide or glycine), dissolving, and then putting into an incubator at 37 ℃ for incubation for 5 minutes to serve as a printing reagent 2;
4) respectively connecting the printing reagents 1 and 2 with different nozzles of a biological printer, printing the printing reagents on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain a hollow tubular three-dimensional structure body;
5) and (3) placing the tubular three-dimensional structure in the step 4) in a WE (Sigma, W2895-1MG) culture solution containing 20ng/ml of EGF, replacing the culture medium every 2 days, and culturing for 2-4 days to form a tubular bile duct-like structure.
Wherein, the distance between the spray head and the winding rod in the step 4) is 3-4 mm.
The motor driver for driving the winding rod to rotate has a rotation speed of 0-10000cts/s (preferably 100cts/s, i.e. one rotation in 100 seconds), and the motor driver for driving the printing nozzle to translate has a rotation speed of 0-10000cts/s (preferably 80cts/s, i.e. one rotation in 125 seconds).
In the present invention, fibrin hydrogel can be obtained by reacting fibrinogen with thrombin, and belongs to enzyme-crosslinked hydrogel. The specific mechanism is as follows: after the thrombin is mixed with the fibrinogen, two sections of polypeptides at the tail ends of alpha and beta chains on the fibrinogen are cut off to form fibrin monomers, and the fibrin monomers are automatically crosslinked to form fibrin hydrogel under the action of hydrogen bonds.
Other materials such as fibrin and gelatin mixture, alginic acid and collagen mixture, etc. can be used in the present invention to replace the fibrinogen-thrombin-calcium chloride system.
In a fifth aspect, the present invention provides a method for constructing a tubular bronchial structure, including the following steps:
1) preparation of printing ink
Dissolving gelatin (Sigma-Aldrich, G1890) and sodium alginate (Sigma-Aldrich, A0682) in 0.5% w/v sodium chloride solution to form 15% gelatin solution and 4% sodium alginate solution, respectively; mixing 600 mu L of gelatin solution and 400 mu L of sodium alginate solution, and keeping the temperature of the obtained mixed solution at 37 ℃ for 20 minutes to obtain printing ink;
2) preparation of printed cells
Respectively culturing human lung bronchial epithelial cells and human fetal lung fibroblasts by adopting an H-DMEM (Hyclone, SH30022.01) medium containing 10% FBS; when the cells grow and are spread on 80-90% of the bottom of the dish, digesting the cells by using an enzyme solution containing 0.04% of EDTA and 0.25% of pancreatin (TargetMol, T0517-50mg), carrying out passage according to the proportion of 1:6, and replacing a culture solution every other day; culturing 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 density of 6X 105Cell culture fluid per ml;
3) and (3) connecting the printing ink of 1) and the cell culture solution of 2) with different nozzles of a biological printer, printing the printing ink and the cell culture solution on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain the hollow tubular bronchial structure.
Wherein, the distance between the spray head and the winding rod in the step 3) is 3-4 mm.
The motor driver for driving the winding rod to rotate has a rotation speed of 0-10000cts/s (preferably 100cts/s, i.e. one rotation in 100 seconds), and the motor driver for driving the printing nozzle to translate has a rotation speed of 0-10000cts/s (preferably 80cts/s, i.e. one rotation in 125 seconds).
The invention provides a tubular tissue structure and a method for constructing the same. The method forms a tubular structure with controllable structure and cell/material composition by a horizontal wound cell 3D printing technique. The tubular structure is similar to the shape and the size of a human body vessel, has mechanical and biological properties meeting the requirements of a human body, and has clinical application potential. The tubular tissue-like structure provided by the invention can be used for the aspects of drug detection, tissue engineering, regenerative medicine, in-vitro physiological model/pathological model/pharmacological model construction, cell structure construction, organoid construction, tissue/organ/human body chip and the like.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
the core components of the invention are a 3D printing nozzle and a winding rod, the printing nozzle is flexible in design, and the printing modes are various. The utilization rate of the printing material is improved, and the configuration requirement of the printing material is reduced. The application range and the combination possibility of the printable material are expanded, so that the applicability is wide, and the construction of the printing material with more complex structure and function is facilitated.
The tubular tissue can be applied to the replacement and bypass operation of various vascular system lesion parts of a human body, and has biocompatibility.
The tubular tissue can be produced in batch, and the construction difficulty of the tubular structure can be reduced, the construction time can be shortened, the construction cost of the tubular tissue can be reduced, and more patients can receive the treatment of tubular diseases.
And fourthly, the tubular tissue can be constructed in a personalized way, and the tubular tissue suitable for different types of vascular systems of human bodies is constructed by mixing and printing ink proportion and cell types according to requirements.
The cells used by the tubular tissue can be autologous cells to construct a tubular structure which can adapt to the physiological environment of a human body and has no immunological rejection reaction and is more suitable for repairing the pathological change part of the vascular system of the human body.
And (VI) in the construction of the tubular tissue, the shape and the size of the winding rod can be changed by changing the operation mode of the printing spray head, and the individualized tubular tissue with the structure size (diameter, thickness, length, curvature, bifurcation and the like) can be constructed.
And (seventhly), the tubular tissue constructed by the invention can be constructed by changing the proportion of printing materials, so that the tubular tissue with directional or staggered protein fiber arrangement has mechanical properties close to human tissues.
The invention (eighthly) adopts the combination of the 3D printing nozzle translation mode and the winding rod unit rotation mode to construct the tubular structure, and the method has the advantages of simple module construction, convenient assembly, detachability, reusability and better operability.
The invention adopts the winding rod unit to construct the tubular structure, and the winding rod unit in the method can be a tubular structure with certain mechanical property and biological property so as to construct the composite tubular tissue with multi-layer structure, mechanical property and biological property.
Drawings
FIG. 1 is a schematic flow chart of a cell printing method according to the present invention.
FIG. 2 shows the structure with cells after printing according to example 1 of the present invention immersed in a culture solution.
FIG. 3 is an optical microscope image of tubular tissue-like structure Day 6 constructed in example 1 of the present invention.
FIG. 4 is a CD31 staining diagram of the tubular tissue-like structure constructed in example 1 of the present invention, which is a schematic diagram of Confocal layer scan three-dimensional reconstruction.
FIG. 5 is a statistical result of the sprouting of endothelial cells in a printed structure according to example 1 of the present invention. The length of cell budding per unit area is shown for different periods of time in culture.
FIG. 6 is a CK19 immunostaining pattern of biliary epithelial progenitor cells in the tubular cholangioid structure in example 2 of the present invention.
Detailed Description
The following examples are intended 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 commercially available products.
The invention utilizes the 3D printing nozzle to accurately control trace substances and crosslinking parameters and the horizontal rotating winding type receiving device to complete the cell three-dimensional printing technology of immediately printing and constructing the tubular structure.
As shown in fig. 1, the three-dimensional cell printing method for constructing a tubular structure of the present invention comprises the following steps:
1) preparation of printing platform
The tubular structure constructed by the present invention is mainly dependent on the construction of the wrap printing apparatus. The design of the winding type printing equipment is mainly divided into two aspects, namely a winding rod capable of performing accurate rotary motion and a printing sprayer capable of performing accurate translation, wherein the translation direction of the printing sprayer is parallel to the winding rod and is positioned in the same vertical direction. Meanwhile, the winding rod and the printing nozzle are controlled by an independent motor driving system, so that the speed of the winding rod and the speed of the printing nozzle are independent and adjustable.
2) Preparation of printing inks (bioprinting materials, printing substrates)
Prepare the printing ink, set the ink dispensing scheme (e.g., but not limited to, composition, volume ratio, etc.). Cells for printing were prepared. The print head is driven by a power driving system (in a pneumatic manner, but not limited to) to eject the print ink.
3) Print parameter setting
And starting a printing mode and controlling a printing process. According to the size (diameter, thickness and length) of the printing tubular tissue structure, the jet speed of printing ink in the 3D printing nozzle is set, the running speed of the winding rod is set, and the translation running speed of the printing nozzle is set. The printing ink in the 3D printing nozzle is pushed out by the nozzle and begins to be attached to the winding rod under the action of gravity. At the same time, the winding rod is rotated in real time, whereby the filamentary compound extruded by the nozzle starts to wind along the winding rod. Meanwhile, the translation of the printing nozzle and the rotation of the winding rod are combined to complete the printing of the tubular structure tissue with specific size.
4) Printed product detection and parameter correction
And taking down the preliminarily printed tubular structure, and detecting the shape and the structure size of the tubular structure. The printing parameters are corrected against their difference from the target tubular structure. And (5) repeating the steps 1-4 to print again.
5) Print the finished product and cultivate
And printing the complete tubular structure with the corresponding length by controlling the printing time. The intact tubular tissue is placed in culture medium for long-term dynamic culture.
6) Detecting the physical and chemical properties and biological properties of tubular tissues
After the tubular tissue is cultured in the culture solution for a period of time, the tubular tissue is taken out for the detection of mechanical property and biological property.
In a preferred embodiment, the printing platform in step 1) may adopt various construction forms, such as a frame type, a ceiling type and other assembly construction forms, to form a printing platform with stable overall structure and convenient operation, so as to ensure stable printing process and reduce printing errors.
In a preferred embodiment, the 3D printing nozzle in step 1) may be in the form of various nozzles, such as a single-axis hollow nozzle, a multiple-axis hollow nozzle, and a microfluidic chip nozzle based on the microfluidic principle
In a preferred embodiment, the printing nozzles in step 1) may be arranged in a plurality of numbers to complete the printing of single-layer and multi-layer tubular structures.
In a preferred embodiment, the winding rod in step 1) may be changed into a clamping mode with a certain inclination angle with the horizontal direction to print a tubular structure with gradually changed thickness.
In a preferred embodiment, the winding rod in step 1) may be replaced by a tubular structure which is pre-cultured by tissue engineering and has certain structural strength and biological activity, so as to construct a composite tubular tissue-like structure with multilayer biological characteristics.
In a preferred embodiment, the winding rod in step 1) can be sleeved in the lumen structure body to construct a composite tubular tissue-like structure with a multi-layer lumen structure.
In a preferred embodiment, the printing nozzle and the winding rod in step 1) are combined in a moving mode, the printing nozzle can move in a translational mode, and the winding rod is fixed and rotated. Or the printing nozzle is fixed and the winding rod rotates to advance. Or both, completing the printing of the tubular structure.
In a preferred embodiment, the print head in step 1) may be configured to reciprocate, so as to print single-layer and multi-layer tubular structures.
In a preferred embodiment, the rotating motion of the winding rod in step 1) can change the rotating direction of the winding rod, or alternatively change the rotating direction with time, so as to construct tubular tissues with different fiber arrangement modes or uneven thickness.
In a preferred embodiment, the rotation speed of the winding rod in the step 1) can be self-regulated, and the translation speed of the printing nozzle can be self-regulated. The two speeds can be independently combined to realize the printing of tubular structures with different structures and sizes.
In a preferred embodiment, the rotating speed of the winding rod and the translational speed of the print nozzle in the step 1) can be self-regulated. The adjusting mode can be realized by controlling the working state of the driving motor or increasing a speed conversion joint and the like.
In a preferred embodiment, in step 1), the printing nozzle may adopt three-dimensional mapping software (such as, but not limited to, Solidworks) to design a three-dimensional structure diagram of the 3D printing nozzle; the existing material (such as but not limited to PDMS, PMMA) is used for processing (such as but not limited to casting) and forming, but not limited to this.
In a preferred embodiment, one or more of glass, resin, etc. may be used for the winding rod in step 1). The cross-sectional configuration may be one or more of circular, elliptical, or polygonal. The diameter may be a single diameter or vary with the axial direction. The central line can be a straight line, a curve, or the like. The printing of tubular structures with different configurations is completed by different arrangements of the winding rods.
In a preferred embodiment, the printing ink material in the step 2) is a mixed solution of a temperature-sensitive material with good cell compatibility and biocompatibility and/or other biological materials; wherein, the biological material can adopt one or more natural biological materials and/or artificial biological materials.
In some embodiments, the natural biomaterial used in the printing ink in step 2) is at least one of the following: gelatin, gelatin derivatives, alginates (such as sodium alginate), alginate derivatives, cellulose derived materials, agar, matrigel, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derived materials, hyaluronic acid derivatives, chitosan derivatives, DNA hydrogel materials, layer-bound proteins, fibronectin, fibrin, fibroin, silk fibroin derivatives, more preferably fibrin derivatives.
In some embodiments, the synthetic biomaterial used in the printing ink of step 2) is at least one of the following materials: polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid, polylactic-co-glycolic acid, polyhydroxy acid, polylactic-co-glycolic acid, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate, polyethylene oxide, preferably polylactic acid or lactic-co-glycolic acid.
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 pericytes, which may be obtained by extraction from a tissue, or may be differentiated from stem cells, preferably vascular endothelial cells and mesenchymal stem cells.
In a preferred embodiment, in the step 3), the printing ink may complete (but not limited to) the reaction before, during and after the pushing action of the printing head, so as to form a filamentous polymer for winding, thereby completing the tubular tissue with the fiber structure.
In some embodiments, the physicochemical and biological properties measured in step 6) can be determined by using experimental methods such as uniaxial tensile test and immunofluorescence test (but not limited thereto).
In a preferred embodiment (example 1), as shown in FIG. 2, the culture medium used for tubular tissue culture is a medium that maintains one or more of a fixed tissue configuration, stability and structural or functional enhancement.
In a preferred embodiment (embodiment 1), as shown in fig. 4, for detecting the shape and size structure of the tubular tissue, detection means such as (but not limited to) an optical microscope and a scanning electron microscope may be applied.
EXAMPLE 1 construction of fibrin tubular vascular-like Structure
1. Print platform preparation
The stainless steel alloy frame structure is constructed, the 3D printing nozzle and the winding rod are assembled in the frame, a motor control system is debugged, a motion system is constructed, the 3D printing nozzle can move in a translation mode, and the winding rod can move in a rotation mode. In the installation, guarantee that 3D prints the shower nozzle and settles directly over the winding stick, be located same vertical plane with the winding stick. The winding stick is placed horizontally, the rotary motion of the winding stick can be controlled accurately, and the motion of the 3D printing nozzle can be controlled accurately along the axial direction of the winding stick. The platform structural member is all customized from the enterprise, and then a few parts are through purchasing raw and other materials such as panel, drawing design by oneself to entrust professional machining unit and process and obtain.
The embodiment adopts a micro-fluidic chip spray head based on the micro-fluidic principle, and a Solidworks structure design software is applied to design a structure diagram of the micro-fluidic chip. The micro-fluidic flow channel adopts a Y + S shape, so that on one hand, the distance traveled by the mixed liquid is increased, more change space is reserved for the pushing speed, and on the other hand, the S-shaped channel is also beneficial to the uniform mixing of the mixed liquid. The round angle treatment is carried out at the convergence inlet of the mixing channel, so that bubbles are prevented from being generated at the position due to sharp corners when liquid flows. In order to avoid influencing the printing performance of the material, the surface of the nozzle material or the channel is selected from a hydrophilic material or a coating so as to reduce the adsorption of fibrin gel and avoid the problem of channel blockage.
This embodiment has designed detachable PMMA chip and has printed the shower nozzle, presss from both sides the one deck pressure sensitive adhesive between two PMMA to use the fix with screw. Meanwhile, the plastic connector is used, the glue-sealed steel needle is used as a guide, and the PVC pipe is used as an injection pipe, so that the PVC pipe has a smoother surface and can effectively avoid liquid seepage.
The winding rod used for the winding type rotation motion is a glass rod with a certain length and diameter.
2. Preparation of printing ink (bioprinting Material)
The printing ink can be purchased through commercial routes and can also be prepared according to actual needs, the tubular fibrin vascular structure is printed, and a printing material (printing ink) is prepared before the tubular fibrin vascular structure is printed, wherein the preparation process comprises the following specific steps:
1) preparation of printing ink for Main Material (bovine fibrinogen and Thrombin)
The bovine fibrinogen needs to be stored in a refrigerator for a long time at the temperature of 20 ℃ below zero and is prepared for use when used, when the bovine fibrinogen is prepared, a weighing balance is sprayed with alcohol and then moved into a super clean bench, ultraviolet irradiation is used for sterilization for 30 minutes, 0.02g of powder is mainly used for printing each time, a tube containing 0.02g of fibrinogen is taken out each time and is dissolved in 500 mul of DMEM/F-12HEPES culture solution, and the DMEM in the culture solution is ensured to be completely dissolved without precipitation.
The thrombin is firstly divided into a plurality of EP tubes by PBS, 1ml of each tube contains 100U and is stored at minus 20 ℃, only one tube is needed for preparing a batch of materials, DMEM/F-12HEPES culture solution is also used for diluting the batch of materials into mother liquor with 5ml of 20U/ml, the mother liquor is wrapped with tinfoil paper and is stored in a refrigerator at 4 ℃ in a dark place, and the batch of mother liquor is printed for a plurality of times. In each printing, a new EP tube is firstly taken, anhydrous calcium chloride particles with the mass of about 0.12g are added into the new EP tube, 500 mu l of thrombin mother liquor is added into the new EP tube to blow and beat the mixture until the anhydrous calcium chloride particles are completely dissolved, and the prepared solution is also placed into an incubator at 37 ℃ for 5 minutes.
The final concentrations used in this example were 20mg/ml fibrinogen, 10U/ml thrombin, 120mM calcium chloride, and the material was aspirated using a 1ml syringe before printing, taking care to minimize the aspiration of air bubbles, and after aspiration the syringe was gently tapped to eliminate air bubbles.
2) Preparation of cells in printing inks
The cells used in this example were HUVEC, human umbilical vein Endothelial cells, cultured using EBM-2Endothelial Growth basic Medium (Lonza), the generation before printing is the fourth generation, before printing, the T75 culture bottle is washed by PBS, digesting in an incubator at 37 ℃ for 2 minutes by using 3ml of 0.25% Trypsin-EDTA (thermal Fisher), adding 6 ml of EBM culture solution to terminate digestion after observing under a mirror, placing in a centrifuge to centrifuge, processing for 3 minutes by using 1000rpm, then taking out the supernatant, adding 1ml of culture solution to resuspend the cells, counting by using a cell counting plate, the cell concentration can be obtained and the total cell number is estimated, the solution amount of the required cell number is taken out after counting, the centrifugation is carried out again, the parameters are the same as the previous step, and 500 mul of fibrinogen solution prepared previously is added after the supernatant fluid is removed to prepare the concentration of 4 multiplied by 10.6cells/ml of cell-containing fibrinogen solution.
3. Print settings
All transfusion work in the printing process is mainly finished by using a Baodingshen SPLab02 injection pump, before each printing, deionized water is firstly used for perfusing a flow channel, 50 mul of the flow channel is washed at the flow rate of 5 mul/min, on one hand, the flow channel is used for washing before use, on the other hand, the flow channel is also used for filling liquid, so that the resistance can be reduced when materials are added, and the stable state in the flow channel can be reached as soon as possible. After the washing is finished, the chip is respectively connected with fibrinogen and thrombin tubes and liquid is introduced into the fibrinogen and thrombin tubes, the fibrinogen and thrombin tubes are similarly set to be 5 mul/min, the total volume is generally set to be 400 mul, a small amount of fast-forward keys on a syringe pump can be used, so that the stable state in a flow channel is quickly achieved, when pink liquid bead gel appears below the chip, the printer beam on a spray head frame of the microfluidic chip can be arranged, and the distance between the spray head and the glass tube for winding is about 3-4 mm.
The printer parameter part adjusts the rotating speed of the winding rod to be 100cts/s, namely 100 seconds per turn, and the rotating speed of a motor driver driving the printing nozzle to translate is 80cts/s, namely 125 seconds per turn, so that the tubular tissue without gaps can be stably printed. The tubular blood vessel-like structure with the total length of 6cm and the wall thickness of 2mm can be printed by one-time printing.
4. Printed product detection and parameter correction
And observing and detecting the printed tubular structural body by using an optical microscope and an electronic scanning microscope, correcting printing parameters (the rotation rate of a winding rod, the jet rate and the translation speed of a nozzle of the microfluidic chip and the like), and printing again.
5. Print the finished product and cultivate
The printed fibrin hollow structure is placed in a culture dish rich in culture solution.
6. Vascularization detection of physical and chemical properties and biological properties of tubular tissues
After the fibrin hollow structure body is cultured in a culture solution for a period of time, the fibrin hollow structure body is taken out for detecting the mechanical property and the biological property. And measuring and calculating the mechanical property of the fibrin hollow structure body by using a uniaxial tensile test, and comparing the mechanical property with the characteristics of human blood vessels. The cell survival status was examined by using CCK8 cell proliferation, and the cell proliferation and growth status was examined by immunofluorescence assay (CD31 and DAPI).
Fig. 3 is an optical microscope image of the tubular tissue-like structure Day 6 constructed in the present example. Figure 4 shows that endothelial cells were able to spread well in the printed oriented fibrin to grow and form choroidal structures. FIG. 5 is a statistical result of the sprouting of endothelial cells in the printed structure, and the results show that the sprouting length of endothelial cells increases with the increase of the culture time, indicating that vascularization starts inside the tubular structure.
Example 2 construction of fibrin tubular bile-like duct Structure
1. Print platform preparation
A printing platform for constructing a stainless steel alloy frame structure mainly comprises a printing nozzle and a winding unit for tubular construction. The printing platform and the components thereof are designed by independent drawing, and each component can be customized by an enterprise or processed by entrusting a professional machining unit.
In the embodiment, a microfluidic chip spray head designed based on a microfluidic principle is applied, and a PMMA material is used for constructing the microfluidic chip spray head with a Y + S-shaped flow channel. The sprayer can effectively promote the fusion of the solution and prevent the problem of channel blockage.
The diameter of the glass tube for winding applied in the embodiment is about 0.5cm, which is similar to the diameter of the bile duct of a human body.
2. Preparation of printing ink (bioprinting Material)
The printing ink can be purchased through commercial approaches and can also be prepared according to actual needs, the fibrin tubular bile duct-like structure is printed, and a printing material (printing ink) is prepared before printing, wherein the specific preparation process comprises the following steps:
1) preparation of printing ink for Main Material (bovine fibrinogen and Thrombin)
0.02g of powder of bovine fibrinogen stored at-20 ℃ in a refrigerator was taken out and dissolved in 500. mu.l of DMEM/F-12HEPES culture medium to ensure complete dissolution of DMEM in the culture medium without precipitation.
The thrombin is firstly divided into a plurality of EP tubes by PBS, 1ml of each tube contains 100U and is stored at minus 20 ℃, only one tube is needed for preparing a batch of materials, DMEM/F-12HEPES culture solution is also used for diluting the batch of materials into mother liquor with 5ml of 20U/ml, the mother liquor is wrapped with tinfoil paper and is stored in a refrigerator at 4 ℃ in a dark place, and the batch of mother liquor is printed for a plurality of times. In each printing, a new EP tube is firstly taken, anhydrous calcium chloride particles with the mass of about 0.12g are added into the new EP tube, 500 mu l of thrombin mother liquor is added into the new EP tube to blow and beat the mixture until the anhydrous calcium chloride particles are completely dissolved, and the prepared solution is also placed into an incubator at 37 ℃ for 5 minutes.
The final concentrations used in this example were 20mg/ml fibrinogen, 10U/ml thrombin and 120mM calcium chloride.
2) Preparation of cells in printing inks
The cells used in this example were human bile duct epithelial progenitor (CP).
hPSCs cell culture: taking a proper amount of hPSCs cells for recovery culture for selection. On the first day, the pluripotent hepatocytes hPSCs were replated with culture medium enriched with activin A (100ng/ml), bFGF (80ng/ml), BMP-4(10ng/ml), LY294002 (10. mu.M) and CHIR99021 (3. mu.M), and cultured overnight at 37 ℃.
Differentiation and culture of hPSCs into DE: the following day, the culture medium was replaced with CDM-PVA supplemented with activin A (100ng/ml), bFGF (80ng/ml), BMP-4(10ng/ml) and LY294002 (10. mu.M). The culture was carried out overnight at 37 ℃. On the third day, the culture medium was replaced with RPMI/B27 supplemented with activin A (100ng/ml) and bFGF (80ng/ml) in a new configuration.
③ differentiation of DE into FP cells: on days 4-6, the old culture medium was replaced with freshly prepared RPMI/B27 supplemented with activin A (50 ng/ml). On day 7,8, the medium was replaced with freshly prepared RPMI/B27 supplemented with activin A (50 ng/ml).
(iv) differentiation of FP cells into HB cells: on days 9-12, the medium was replaced with freshly prepared RPMI/B27 medium containing SB-431542 (10. mu.M) and BMP-4(50 ng/ml). Differentiation of HB hepatic progenitors was detected by expression of HNF4A, AFP and TBX3 and flow analysis. Realizing the differentiation of FP cells to HB cells.
Differentiation of HBs cells into CPs: on days 13-16, the differentiation of biliary epithelial progenitor cells was examined by Sox9 expression using freshly prepared RPMI/B27 medium containing FGF10(50ng/ml), activinA (50ng/ml) and retinic acid (3. mu.M) instead of the medium. Ensuring the differentiation of the hepatic progenitor cells to the biliary epithelial progenitor cells.
Sixthly, washing the cells by using PBS, adding cell digestive juice, and storing for 20 minutes at 37 ℃. The cells were separated from the bottom plate and collected using a pipette. The cells were transferred to a 15 ml tube, gently insufflated and resuspended 2-3 times, and the cells were separated into small clumps using a 1000 microliter pipette. The plates were rinsed with RPMI/B27 medium and transferred to 15 ml tubes. Centrifuge for 3 minutes at room temperature. The supernatant was aspirated and the cells were resuspended in 6 ml of RPMI/B27. Centrifuge for 3 minutes at room temperature and aspirate the supernatant. The cells were resuspended in a pre-prepared 50% matrix gel containing EGF (20ng/ml) and Rho kinase inhibitor Y-27632(10 μm) and mixed well.
Counting with cell counting plate to obtain cell concentration and estimate total cell number, taking out the solution amount of the required cell number after counting, centrifuging again, removing supernatant, adding 500 μ l fibrinogen solution to obtain the final product with concentration of 4 × 106cells/ml of cell-containing fibrinogen solution.
3. Print settings
The SPLab02 injection pump is used for filling and pressure driving of printing ink, and deionized water is used for filling the flow channel before printing each time, so that the resistance can be reduced when materials are added, and the stable state in the flow channel can be achieved as soon as possible. After the washing, the chip is respectively connected with the fibrinogen and thrombin tubes and the liquid is introduced into the fibrinogen and thrombin tubes, the fibrinogen and thrombin tubes are also set to be 5 mul/min, the total volume is generally set to be 400 mul, and the injection pump is adjusted to ensure that the flow channel quickly reaches a steady state. When pink liquid bead gel appears below the chip, the distance between the spray head and the winding glass tube is about 3-4 mm.
The printer parameter part is adjusted to ensure that tubular tissues without gaps can be stably printed.
4. Printed product detection and parameter correction
And observing and detecting the printed tubular structural body by using an optical microscope and an electronic scanning microscope, correcting printing parameters (the rotation rate of a winding rod, the jet rate and the translation speed of a nozzle of the microfluidic chip and the like), and printing again.
5. Print the finished product and cultivate
The printed fibrin bile duct structures were placed in freshly prepared WE medium of EGF (20ng/ml) and the medium was replaced every 2 days. Organoid tissues are formed in 2-4 days of culture.
6. Bile duct-like detection of physicochemical and biological properties of tubular tissues
After the fibrin hollow structure body is cultured in a culture solution for a period of time, the fibrin hollow structure body is taken out for detecting the mechanical property and the biological property. The differentiation of cholangiocarcinoma-like cells was examined by expression of CK7, ensuring that it could be observed in > 75% of the cells. The expression of CK19 is detected by immunofluorescence staining, and the growth condition of bile duct epithelial cells is characterized. The alkaline phosphatase staining expression and the activity of glutamyltranspeptidase for characterizing the bile duct epithelial cell function were examined. The CK19 immunofluorescent staining pattern (FIG. 6) shows positive expression of CK19, indicating that the growth state of biliary epithelial cells is good and a tubular structure is generated.
Example 3 construction of tubular bronchial-like Structure
1. Print platform preparation
A printing platform for constructing a stainless steel alloy frame structure mainly comprises a printing nozzle and a winding unit for tubular construction. The printing platform and the components thereof are designed by independent drawing, and each component can be customized by an enterprise or processed by entrusting a professional machining unit.
The embodiment applies the microfluidic chip spray head designed based on the microfluidic principle and adopts PMMA material to construct the microfluidic chip spray head with a single flow channel. The spray head can accurately control the flow of the solution.
The diameter of the glass tube used for winding in this example is about 1cm, which is similar to the diameter of a human bronchus.
2. Preparation of printing ink (bioprinting Material)
The printing ink can be purchased through commercial routes and can also be prepared according to actual needs, the tubular bronchial structure is printed in the embodiment, and the printing material (printing ink) is prepared before printing, and the specific preparation process comprises the following steps:
1) preparation of printing ink for Main Material (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% strength gelatin solution and a 4% strength sodium alginate solution, respectively.
2) Preparation of cells in printing inks
The cells used in this example were human lung bronchial epithelial cells (Beas-2B) and human fetal lung fibroblasts (MRC-5).
Cell culture: the culture was carried out in H-DMEM medium (Hyclone, SH30022.01) (containing 10% FBS). When the cells grew to about 80% of the bottom of the dish, they were digested with 0.25% pancreatin (TargetMol, T0517-50mg) (containing 0.04% EDTA), passaged at a ratio of 1:6, and the culture medium was changed every other day.
3. Print settings
600. mu.L of gelatin solution and 400. mu.L of sodium alginate solution were incubated at 37 ℃ for 20 minutes and gently mixed to prepare a matrix material. Beas-2B and MRC-5 cells (cell density 6X 10)5One/ml, the ratio of the two is 5: 1).
The SPLab02 injection pump is used for filling and pressure driving of printing ink, and deionized water is used for filling the flow channel before printing each time, so that the resistance can be reduced when materials are added, and the stable state in the flow channel can be achieved as soon as possible. After the washing is finished, the chip is connected with the mixed solution and is filled with the solution, the concentration is also set to be 5 mul/min, the total concentration is generally set to be 400 mul, and the injection pump is adjusted to ensure that the flow channel quickly reaches a stable state. The distance between the spray head and the glass tube for winding is about 3-4 mm.
The printer parameter part is adjusted to ensure that tubular tissues without gaps can be stably printed.
4. Printed product detection and parameter correction
And observing and detecting the printed tubular structural body by using an optical microscope and an electronic scanning microscope, correcting printing parameters (the rotation rate of a winding rod, the jet rate and the translation speed of a nozzle of the microfluidic chip and the like), and printing again.
5. Print the finished product and cultivate
Placing the printed bronchial structure in 5% CO2And cultured in an incubator at 37 ℃ and replaced every 1 to 2 days in a fresh prepared H-DMEM medium (containing 10% FBS).
6. Bronchiolization-like detection of physicochemical and biological properties of tubular tissues
After the bronchus-like hollow structure body is cultured in a culture solution for a period of time, the bronchus-like hollow structure body is taken out and the detection of mechanical property and biological property is carried out. After the bronchial-like structure is constructed and grows for 7 days, HE staining shows the phenomenon of cell connection growth; the broad spectrum CK and Vimentin staining both have positive results, which indicates that the cell activity is better.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The method for constructing the tubular tissue-like structure is characterized by comprising the following steps of:
A. preparing printing ink;
B. culturing cells for printing in vitro to obtain a cell culture solution;
C. respectively connecting the printing ink and the cell culture solution with different nozzles of a biological printer, printing the printing ink and the cell culture solution on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain a hollow tubular tissue structure;
wherein, the printing ink is made of temperature-sensitive material and/or biological material with good cell compatibility and biocompatibility; the biological material adopts one or more natural biological materials and/or artificially synthesized biological materials;
the natural biomaterial is selected from at least one of gelatin, gelatin derivatives, alginate derivatives, cellulose-derived materials, agar, matrigel, collagen derivatives, amino acids, amino acid derivatives, proteoglycans, proteoglycan derivatives, glycoproteins and derived materials, hyaluronic acid derivatives, chitosan derivatives, DNA hydrogel materials, layer-connected proteins, fibronectin, fibrin, silk fibroin and silk fibroin derivatives;
the artificial synthetic biomaterial is at least one selected from polypropylene, polystyrene, polyacrylamide, polylactide, polyglycolide, polylactic acid-glycolic acid copolymer, polyhydroxy acid, polylactic acid-alkyd copolymer, polydimethylsiloxane, polyanhydride, polyacid ester, polyamide, polyamino acid, polyacetal, polycyanoacrylate, polyurethane, polypyrrole, polyester, polymethacrylate, polyethylene, polycarbonate and polyethylene oxide.
2. The method according to claim 1, wherein the cells in step B are vascular cells selected from at least one of vascular endothelial cells, vascular endothelial progenitor cells, microvascular endothelial cells, vascular smooth muscle cells, vascular fibroblasts, mesenchymal stem cells, pericytes, preferably vascular endothelial cells and mesenchymal stem cells;
wherein the vascular cells are obtained by extraction from a tissue or are differentiated from stem cells.
3. The method of claim 1, wherein the wound rod is a glass rod; and/or
The distance between the spray head and the winding rod is 3-4 mm; and/or
The rotating speed of the motor driver for driving the winding rod to rotate is 0-10000cts/s, and the rotating speed of the motor driver for driving the printing nozzle to translate is 0-10000 cts/s.
4. A tubular tissue-like structure constructed according to the method of any one of claims 1 to 3.
5. The method for constructing the tubular vascular structure is characterized by comprising the following steps of:
1) dissolving 0.02g bovine fibrinogen in 500 mul DMEM/F-12HEPES culture solution to obtain fibrinogen solution;
2) HUVEC (human umbilical vein Endothelial cells) are cultured in vitro by using EBM-2Endothelial Growth basic Medium and subcultured, the HUVEC is cultured to the fourth generation before printing, PBS is used for washing the cells, then Trypsin-EDTA is added into a culture bottle, the HUVEC is digested for 2 minutes in an incubator at 37 ℃, and then EBM culture solution is added to stop digestion; put into a centrifuge forCentrifuging, removing supernatant, resuspending the cells with culture medium, counting, centrifuging again, removing supernatant, adding the fibrinogen solution of step 1) to the cell precipitate to obtain 4 × 106cells/ml of fibrinogen solution containing cells as printing reagent 1;
3) preparing 20U/ml thrombin mother solution by using DMEM/F-12HEPES culture solution; before printing, adding 500 mu l of thrombin mother liquor into 0.12g of anhydrous calcium chloride, dissolving, and then putting into an incubator at 37 ℃ for incubation for 5 minutes to serve as a printing reagent 2;
4) and respectively connecting the printing reagents 1 and 2 with different nozzles of a biological printer, printing the printing reagents on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain the hollow tubular vascular-like structure.
6. The method as claimed in claim 5, wherein the distance between the spray head and the winding rod in step 4) is 3-4 mm; and/or
The rotating speed of the motor driver for driving the winding rod to rotate is 0-10000cts/s, and the rotating speed of the motor driver for driving the printing nozzle to translate is 0-10000 cts/s.
7. The method for constructing the tubular bile duct-like structure is characterized by comprising the following steps of:
1) dissolving 0.02g bovine fibrinogen in 500 mul DMEM/F-12HEPES culture solution to obtain fibrinogen solution;
2) preparation of printing agent 1:
hPSCs cell culture: taking a proper amount of hPSCs cells for recovery culture, wherein the culture solution is as follows: 100ng/ml activin A, 80ng/ml bFGF, 410 ng/ml BMP-410 ng/ml LY 29400210. mu.M and CHIR 990213. mu.M, and culturing overnight at 37 ℃;
differentiation and culture of hPSCs into DE cells: the next day, the culture solution of (i) was replaced with CDM-PVA culture solution supplemented with activin A100 ng/ml, bFGF80ng/ml, BMP-410 ng/ml and LY 29400210. mu.M, and cultured overnight at 37 ℃; on the third day, the culture solution of RPMI/B27 added with activin A100 ng/ml and bFGF80ng/ml is used for replacing the old culture solution;
③ differentiation of DE cells into FP cells: on days 4-6, replacing the old culture medium with RPMI/B27 supplemented with activin A50 ng/ml; on days 7-8, replacing the old culture medium with RPMI/B27 supplemented with activin A50 ng/ml;
(iv) differentiation of FP cells into HB cells: on days 9-12, the old medium was replaced with RPMI/B27 containing SB-43154210. mu.M and BMP-450 ng/ml; detecting the differentiation of HB cells by measuring the expression of HNF4A, AFP and TBX3 genes and flow analysis;
differentiation of HB cells into CP: on days 13-16, the old culture medium was replaced with RPMI/B27 containing FGF 1050 ng/ml, activin A50 ng/ml and retinic acid 3. mu.M, and the differentiation of CP cells was examined by measuring the expression of Sox9 gene;
sixthly, washing the CP cells by PBS, adding cell digestive juice, incubating for 20 minutes in an incubator at 37 ℃, and collecting the cells by a pipette; transferring the cells to RPMI/B27 culture solution, resuspending the cells, centrifuging at room temperature for 3 min, discarding the supernatant, resuspending the cells in 50% Matrigel matrix gel containing EGF20ng/ml and Rho kinase inhibitor Y-2763210 μm, counting, centrifuging again, discarding the supernatant, adding the fibrinogen solution of step 1) to the cell pellet to obtain 4 × 106cells/ml of fibrinogen solution containing cells as printing reagent 1;
3) preparing 20U/ml thrombin mother solution by using DMEM/F-12HEPES culture solution; before printing, adding 500 mu l of thrombin mother liquor into 0.12g of anhydrous calcium chloride, dissolving, and then putting into an incubator at 37 ℃ for incubation for 5 minutes to serve as a printing reagent 2;
4) respectively connecting the printing reagents 1 and 2 with different nozzles of a biological printer, printing the printing reagents on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain a hollow tubular three-dimensional structure body;
5) and (3) placing the tubular three-dimensional structure in the step 4) into a WE culture solution containing 20ng/ml of EGF, replacing the culture medium every 2 days, and culturing for 2-4 days to form a tubular bile duct-like structure.
8. The method as claimed in claim 7, wherein the distance between the spray head and the winding rod in step 4) is 3-4 mm; and/or
The rotating speed of the motor driver for driving the winding rod to rotate is 0-10000cts/s, and the rotating speed of the motor driver for driving the printing nozzle to translate is 0-10000 cts/s.
9. The method for constructing the tubular bronchial structure is characterized by comprising the following steps of:
1) preparation of printing ink
Dissolving gelatin and sodium alginate in 0.5% w/v sodium chloride solution to form 15% gelatin solution and 4% sodium alginate solution; mixing 600 mu L of gelatin solution and 400 mu L of sodium alginate solution, and keeping the temperature of the obtained mixed solution at 37 ℃ for 20 minutes to obtain printing ink;
2) preparation of printed cells
Respectively culturing human lung bronchus epithelial cells and human fetal lung fibroblasts by adopting a H-DMEM culture medium containing 10% FBS; when the cells grow and are spread to 80-90% of the bottom of the dish, digesting the cells by using enzyme solution containing 0.04% of EDTA and 0.25% of pancreatin, carrying out passage according to the proportion of 1:6, and replacing culture solution every other day; culturing 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 density of 6X 105Cell culture fluid per ml;
3) and (3) connecting the printing ink of 1) and the cell culture solution of 2) with different nozzles of a biological printer, printing the printing ink and the cell culture solution on a winding rod together to form a seamless tubular tissue, and then removing the winding rod to obtain the hollow tubular bronchial structure.
10. The method as claimed in claim 9, wherein the distance between the spray head and the winding rod in step 3) is 3-4 mm; and/or
The rotating speed of the motor driver for driving the winding rod to rotate is 0-10000cts/s, and the rotating speed of the motor driver for driving the printing nozzle to translate is 0-10000 cts/s.
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| CN107164318A (en) * | 2017-07-19 | 2017-09-15 | 叶川 | Method and hollow form vascularised heart based on 3D biometric print technique construction hollow form vascularised hearts |
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| CN112843334A (en) * | 2021-01-13 | 2021-05-28 | 东华大学 | Bionic trachea constructed by three-dimensional printing composite aerogel and preparation method thereof |
| CN112843334B (en) * | 2021-01-13 | 2022-07-08 | 东华大学 | Bionic trachea constructed by three-dimensional printing composite aerogel and preparation method thereof |
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| CN117717434A (en) | 2024-03-19 |
| CN117679205A (en) | 2024-03-12 |
| CN112206074B (en) | 2024-01-02 |
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