WO2016054847A1 - Bionic structure containing channels and electromagnetic force training device and method therefor - Google Patents

Bionic structure containing channels and electromagnetic force training device and method therefor Download PDF

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Publication number
WO2016054847A1
WO2016054847A1 PCT/CN2014/089771 CN2014089771W WO2016054847A1 WO 2016054847 A1 WO2016054847 A1 WO 2016054847A1 CN 2014089771 W CN2014089771 W CN 2014089771W WO 2016054847 A1 WO2016054847 A1 WO 2016054847A1
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Prior art keywords
channel
cells
cell
pulsation
bionic structure
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PCT/CN2014/089771
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French (fr)
Chinese (zh)
Inventor
王小红
许雨帆
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清华大学
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Priority claimed from CN201410535181.8A external-priority patent/CN104306083B/en
Application filed by 清华大学 filed Critical 清华大学
Publication of WO2016054847A1 publication Critical patent/WO2016054847A1/en

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    • A—HUMAN NECESSITIES
    • 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
    • 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
    • A61F2/02—Prostheses implantable into the body

Definitions

  • organ transplantation technology has always faced a series of problems such as immune rejection, donor shortage, organ distribution, and ethics. According to statistics, more than 1.5 million patients need organ transplants each year in China, but the supply-demand ratio is less than 1:100 [Langer R. Tissue Engineering, 2007, 13(1): 1-2]. Tissue Engineering and Organ Manufacturing use a combination of engineering and medicine to create a new solution to this problem from the organizational and organ levels.
  • the biomimetic structure of artificial tissue or organ can be used to implant the body to repair tissue defects, replacing organ function; or as an extracorporeal device, temporarily replacing organ function [Cao Yilin et al., Journal of Clinical Surgery, 2007, 15(1): 40- 41].
  • tissue organs constructed by tissue engineering methods have been clinically applied, but these applications are mostly confined to tissues such as cartilage and epithelium. These tissues are simple in structure, single in composition, and limited in size.
  • the main problem is to construct functional multi-organ substitutes while constructing functional tissues and scaffold structures. These functions mainly include circulatory system, nervous system and immune system.
  • a channel-containing biomimetic structure characterized in that the biomimetic structure comprises a structural body and at least one channel, the channel is distributed in the structural body; the structural body is a mixture of a natural polymer hydrogel and a cell.
  • the cell concentration is 10-10 8 /mL, and the cell is at least one of an embryonic stem cell, an adult stem cell, an adult cell, a cancer cell, and an induced pluripotent stem cell;
  • the channel is a single channel or a branch channel, and the channel is One or more combinations of through-holes at both ends, blind holes at both ends, and one-end through-hole structures at one end; the positional relationship between the channels is intersecting, parallel, collinear or different; the outer wall of the channel and the inner wall of the channel Or cells are distributed in the pores of the channel, and the cells are at least two of the seed cells of the nervous system, the seed cells of the vascular system, and the seed cells of the immune system.
  • the cross section of the channel is circular, elliptical, polygonal or irregular geometrical, and the channel cross-sectional area is 100 ⁇ m 2 -1 cm 2 .
  • the channel-containing biomimetic structure is characterized in that: the nervous system seed cells are at least one of neurons and glial cells; and the blood vessel seed cells are endothelial cells, smooth muscle cells and adipose stem cells. At least one of the immune system seed cells is at least one of lymphocytes and innate immune cells.
  • the natural polymer hydrogel of the present invention is at least one of sodium alginate, collagen, matrigel, dextrose, chitosan, gelatin and fibrinogen, and the mass concentration of the hydrogel is 0.1 to 20%.
  • the natural polymer hydrogel is compounded with at least one of a cell cryopreservative, a cell growth factor, a drug, an anticoagulant, and a magnetic nanoparticle;
  • the cell cryopreservative is dimethyl sulfoxide, glycerin And at least one of dextrose;
  • the cell growth factor is at least one of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, human platelet derived growth factor, and transforming growth factor;
  • the drug is at least one of an antitumor drug and a virus vaccine;
  • the anticoagulant is at least one of heparin and paclitaxel;
  • the magnetic nanoparticles are ferrite particles, metal particles and nitriding
  • the present invention provides an electromagnetic force training device for a channel-containing bionic structure, characterized in that the device comprises an electric field generating system, a magnetic field generating system, a sample stage and a fixed platform; the magnetic field generating system and the sample stage are fixed at a fixed position On the platform; the electric field generating system includes a positive electrode and a negative electrode; the sample stage is provided with a guide rail, and the positive electrode and the negative electrode are respectively mounted on the guide rail through a slider; the sample stage is located in the magnetic field generating system; the magnetic field generating system
  • the sub-assembly includes a bracket and a rotatable ring mounted in the hollow structure of the bracket, and a detachable S pole and N pole are radially symmetrically arranged on the rotatable ring.
  • the present invention also provides a method of training a biomimetic structure using an electric field of the above apparatus, characterized in that the method comprises the following steps:
  • the current is greater than 0, less than or equal to 50 mA; the voltage is greater than 0, less than or equal to 50 V
  • the current direction is alternating between AC, DC or both;
  • the bionic structure After completing the training of the electric field on the bionic structure containing the channel, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  • the bionic structure After completing the training of the magnetic field on the channel-containing bionic structure, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  • the present invention also provides a method of training a biomimetic structure using an electromagnetic composite field of the above apparatus, characterized in that the method comprises the following steps:
  • the pulsation system motor is connected to the pulsation system rail-slider mechanism by a crank;
  • the guide rail-slider The mechanism is connected with the culture liquid supply syringe;
  • the bionic structure is connected to the pulsation culture system through the flow guiding tube;
  • the culture liquid is unidirectionally flowed between the draft tube and the bionic structure containing the channel;
  • the liquid flow rate is greater than 0, less than At 30 cm/s, the action of the pulsation-electric field, the pulsation-magnetic field or the pulsation-electromagnetic composite field is performed; after the training of the bionic structure is completed, the bionic structure is cryopreserved, or the in vitro culture is continued or directly used for organ transplantation.
  • the present invention has the following advantages and outstanding technical effects:
  • the invention is based on the existing three-dimensional forming technology such as 3D printing method and rotating combined mold method, and further compounding the cell and polymer gel mixture to prepare a bionic structure of channel, multi-system and multifunctional complex organizer.
  • the channel-containing biomimetic structure encompasses the vascular system, the nervous system, and the immune system, and structurally and functionally mimics the true multi-system state of the body.
  • the electromagnetic force training device of the invention combines the functions of electric field, magnetic field and pulsation culture, and performs in vitro training and mechanical culture on the bionic structure, so that the cells can be positioned layer by layer and arranged in the pipeline structure under the action of the composite field. Peripheral, close to the cell morphology of the real organ duct, the formed structure has good performance in morphology, immunophysiology.
  • an electromagnetic force training device can use an electric field alone, use a magnetic field alone or simultaneously use an electromagnetic field; in addition, the introduction of a pulsation culture system also increases the diversification of device functions.
  • Figure 1 is a schematic diagram of a bionic tissue structure containing channels.
  • FIG. 2a-2i are schematic views of the state of several channel cells; wherein, FIG. 2a is a schematic diagram of a cell located on the outer wall of the channel, FIG. 2b is a schematic view of a cell located on the inner wall of the channel, and FIG. 2c is a view of a cell located in the channel hole.
  • Schematic diagram, Fig. 2d is a schematic diagram of two cells located on the outer wall of the channel
  • Fig. 2e is a schematic diagram of two cells located on the inner wall of the channel
  • Fig. 2f is a schematic diagram of two cells in the channel hole
  • Fig. 2g is a cell located on the outer wall of the channel.
  • 2h is a schematic diagram of a cell located in the channel wall and another cell located in the channel hole.
  • FIG. 2i is a schematic diagram of a cell located on the outer wall of the channel and another cell located on the inner wall of the channel.
  • Fig. 3 is a schematic view showing the formation of a bionic structure containing a channel by a multi-nozzle 3D printing method.
  • 4a and 4b are respectively a schematic view and an exploded view of a bionic structure of a multi-core rotating combined mold forming channel.
  • 5a, 5b and 5c are respectively a schematic view of a spindle-shaped two-channel biomimetic structure, a schematic diagram of a block-shaped cross-channel bionic structure and a block-like parallel channel bionic structure.
  • Figure 6 is a schematic view of an electromagnetic training device.
  • Figure 7a is a schematic diagram of the explosion of the magnetic field generating system
  • Figure 7b is a schematic diagram of the pulsating culture system.
  • Figure 8a shows the bionic structure containing the channel between the metal plate electrodes
  • Figure 8b shows the bionic structure of the channel between the metal wire or the metal probe
  • Figure 8c is a schematic diagram of the movement of the charged particles in the electric field (E).
  • Fig. 9a shows that the bionic structure containing the channel is located between the magnetic field S and the N pole
  • Fig. 9b is a schematic view of the movement of the magnetic particle in the magnetic field (B) (the broken line indicates the direction of the magnetic field rotation).
  • Fig. 10a shows that the bionic structure containing the channel is located between the positive and negative electric fields and the magnetic field S and N
  • Fig. 10b is a schematic diagram of the movement of the charged particles in the composite electromagnetic field (the broken line indicates the direction of rotation of the magnetic field).
  • a channel-containing biomimetic structure includes a structural body 101 and at least one channel distributed in the structural body 101; the structural body is a mixture of a natural polymer hydrogel and a cell.
  • the cell concentration is 10-10 8 /mL, the cell is at least one of an embryonic stem cell, an adult stem cell, an adult cell, a cancer cell and an induced pluripotent stem cell;
  • the channel is a single channel or a branch channel 105, the channel One or more combinations of the through holes 103 at both ends, the blind holes 106 at both ends, and the one end through holes 104 at one end; the positional relationship between the channels is intersecting, parallel, collinear or different;
  • Cells are distributed in the outer wall, the inner wall of the channel or the pores of the channel, and the cells are at least two of the seed cells of the nervous system, the seed cells of the vascular system, and the seed cells of the immune system.
  • the channel has a circular, elliptical, polygonal or irregular geometry and has a channel cross-sectional area of 100 ⁇ m 2 -1 cm 2 .
  • the nervous system seed cell is at least one of a neuron and a glial cell;
  • the blood vessel seed cell is at least one of an endothelial cell, a smooth muscle cell, and an adipose stem cell;
  • the immune system seed cell is a lymphocyte and At least one of innate immune cells.
  • FIG. 2a-2i are schematic views of the state of several channel cells; wherein, FIG. 2a is a schematic diagram of a cell located on the outer wall of the channel, FIG. 2b is a schematic view of a cell located on the inner wall of the channel, and FIG. 2c is a view of a cell located in the channel hole.
  • Schematic diagram Fig. 2d is a schematic diagram of two cells located on the outer wall of the channel, Fig. 2e is a schematic diagram of two cells located on the inner wall of the channel, and Fig. 2f is two thin Schematic diagram of the cell located in the pore of the channel, FIG. 2g is a schematic diagram of another cell located in the channel pore on the outer wall of the channel, and FIG.
  • FIG. 2h is a schematic diagram of another cell located in the channel hole on the inner wall of the channel
  • FIG. 2i A schematic diagram of a cell located on the outer wall of the channel and another cell located on the inner wall of the channel.
  • FIG. 4a and FIG. 4b it is a preparation method of a biomimetic structure.
  • Fig. 3 is a schematic view showing the formation of a bionic structure containing a channel by a multi-nozzle 3D printing method.
  • 4a and 4b are respectively a schematic view and an exploded view of a bionic structure of a multi-core rotating combined mold forming channel.
  • Figures 5a to 5c are schematic views of several biomimetic structures containing multiple channels.
  • 5a, 5b and 5c are respectively a schematic view of a spindle-shaped two-channel biomimetic structure, a schematic diagram of a block-shaped cross-channel bionic structure and a block-like parallel channel bionic structure.
  • the natural polymer hydrogel is at least one of sodium alginate, collagen, matrigel, dextrose, chitosan, gelatin and fibrinogen, and the mass concentration of the hydrogel is 0.1 to 20%. .
  • the natural polymer hydrogel is compounded with at least one of a cell cryopreservative, a cell growth factor, a drug, an anticoagulant, and a magnetic nanoparticle;
  • the cell cryopreservative is dimethyl sulfoxide, glycerin And at least one of dextrose;
  • the cell growth factor is at least one of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, human platelet derived growth factor, and transforming growth factor;
  • the drug is at least one of an antitumor drug and a virus vaccine;
  • the anticoagulant is at least one of heparin and paclitaxel;
  • the magnetic nanoparticles are ferrite particles, metal particles and nitriding At least one of the iron particles.
  • an electromagnetic training device includes an electric field generating system 601, a magnetic field generating system, a sample stage 603, and a fixed platform 605; the magnetic field generating system and sample stage 603 are mounted on a fixed platform 605;
  • the system 601 includes a positive electrode and a negative electrode;
  • the sample stage 603 is provided with a guide rail, and the positive electrode and the negative electrode are respectively mounted on the guide rail through a slider;
  • the sample stage 603 is located in the magnetic field generating system;
  • the magnetic field generating system includes a bracket 602 and a rotatable ring 608, the rotatable ring 608 is mounted in the hollow structure of the bracket 602, and the detachable S pole 606 and the N pole 607 are radially symmetrically arranged on the rotatable ring 608.
  • the device may further include a pulsation culture system 701; the pulsation culture system is mounted on the fixed platform 605; the pulsation culture system 701 includes a pulsation system motor 702, a pulsation system guide-slider mechanism 703, a culture fluid supply injector 704, a check valve 705, a draft tube 706 and a culture fluid bottle 708; the pulsation system motor 702 is connected to the pulsation system rail-slider mechanism 703 by a crank; the pulsation system rail-slider mechanism 703 and the culture fluid supply syringe 704 Connection, see Figure 7b.
  • the positive electrode and the negative electrode are one of a metal plate, a metal wire or a metal probe.
  • the S pole and the N are one of a permanent magnet or an electromagnet.
  • FIG. 8a the channel-containing biomimetic structure is located between the metal plate electrodes
  • FIG. 8b shows that the channel-containing bionic structure is located between the metal wires or the metal probes
  • FIG. 8c is a schematic diagram of the movement of the charged particles in the electric field (E). .
  • the method for training a channel-containing biomimetic structure in an electric field of the device includes the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture liquid containing suspended cells, and placing the electromagnetic force On the sample stage of the training device; b) starting the electric field generating system of the electromagnetic training device, removing the N and S stages of the magnetic field generating system; and making the bionic structure containing the channel in the electric field; the current is greater than 0, less than or equal to 50 mA; It is greater than 0, less than or equal to 50V; the current direction is alternately used by alternating current, direct current or both; c) after completing the training of the electric field on the bionic structure containing the channel, the biomimetic structure is cryopreserved or continuously cultured in vitro or directly used Organ transplantation.
  • Fig. 9a the channel-containing biomimetic structure is located between the magnetic field S and the N pole
  • Fig. 9b is a schematic diagram of the movement of the magnetic particles in the magnetic field (B) (the broken line indicates the direction of the magnetic field rotation).
  • the method for training a channel-containing biomimetic structure in a magnetic field of the device includes the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture liquid containing suspended cells, and placing the electromagnetic force On the sample stage of the training device; b) turning off the electric field generating system of the electromagnetic training device, starting the magnetic field generating system, rotating the S pole and the N pole with the rotatable ring, controlling the magnetic induction intensity to be greater than 0, less than or equal to 5T; c) completing After the magnetic field is trained on the bionic structure containing the channel, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  • Fig. 10a shows that the bionic structure containing the channel is located between the positive and negative electric fields and the magnetic field S and N
  • Fig. 10b is a schematic diagram of the movement of the charged particles in the composite electromagnetic field (the broken line indicates the direction of rotation of the magnetic field).
  • the method for training a channel-containing biomimetic structure in the electromagnetic composite field of the device comprises the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture fluid containing suspended cells, and placing On the sample stage of the electromagnetic force training device; b) simultaneously starting the electric field generating system and the magnetic field generating system of the electromagnetic training device, so that the bionic structure containing the channel is located in the electric field and the magnetic field; the control current is greater than 0, less than or equal to 50 mA; and the voltage is greater than 0, less than or equal to 50V; current direction is alternating between alternating current, direct current or both; controlling magnetic induction intensity is greater than 0, less than or equal to 5T; c) after completing the electric field and magnetic field simultaneously training the bionic structure containing the channel, the bionic structure Store at low temperature, or continue in vitro culture or directly for organ transplantation.
  • the present invention also applies the training method to the pulsation culture system, the steps including the above steps, and the introduction and use of the pulsation culture system: starting a pulsation culture system 701, the pulsation culture system 701 includes a pulsation system motor 702, a pulsation system guide-slider mechanism 703, a culture fluid supply syringe 704, a check valve 705, a draft tube 706, and a culture fluid bottle 708;
  • the system motor 702 is coupled to the pulsation system rail-slider mechanism 703 by a crank; the rail-slider mechanism 703 is coupled to the culture fluid supply injector 704;
  • the biomimetic structure is coupled to the pulsation culture system via a flow conduit 706;
  • the liquid flows in a one-way flow between the draft tube 706 and the bionic structure containing the channel; the liquid flow rate is greater than 0, less than 30 cm/s,
  • Example 1 A pseudo-heart structure containing two channels of blood vessels and nervous systems was prepared by multi-nozzle 3D printing, and the pseudo-heart structure was trained in an electric field.
  • cardiomyocytes are used as host cells, mixed with the configured sodium alginate solution, and the sodium alginate hydrogel solution has a mass volume concentration of 0.5%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
  • vascular endothelial cells and neurons as channel cells; mixing vascular endothelial cells with sodium alginate solution into a second nozzle assembly of a 3D printing device; mixing neurons with cell culture fluid (DMEM), loading Into the third nozzle assembly of the 3D printing device;
  • DMEM cell culture fluid
  • d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of myocardial cells / sodium alginate in the main structure; synchronous or asynchronous control of vascular endothelial cells / sodium alginate channel formation, get vascular system channels; synchronization or Asynchronously control the neurons/DMEM, locate them in the channel pores, and obtain the nervous system channels; and cross-link the sodium alginate solution with CaCl2 to obtain a sodium alginate hydrogel, which is stacked layer by layer to obtain the multi-channel double The simulated heart structure of the channel;
  • the electric field generating device of the electromagnetic training device will be energized, and the control voltage is DC 50V;
  • Example 2 A multi-nozzle 3D printing method was used to prepare a pseudo-renal structure containing three channels of blood vessels, nerves and immune system, and the simulated kidney structure was trained in an electric field.
  • the embryonic kidney cells are used as host cells, mixed with the configured fibrinogen solution, and the fibrinogen solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
  • vascular endothelial cells, smooth muscle cells, neurons, and T lymphocytes as channel cells; mixing vascular endothelial cells and smooth muscle cells with sodium alginate solution, and loading them into a second nozzle assembly of the 3D printing device; Mixing with the sodium alginate solution, loading into the third nozzle assembly of the 3D printing device; mixing the T lymphocytes with the sodium alginate solution into the fourth nozzle assembly of the 3D printing device;
  • the electric field generating device of the electromagnetic training device will be energized, and the control current is DC 50 mA;
  • Example 3 A multi-nozzle 3D printing method was used to prepare a liver-like structure containing five channels of blood vessels, nerves, bile ducts, and immune system, and the liver-like structure was trained in a magnetic field.
  • vascular endothelial cells and B lymphocytes as channel cells (both of which have been mixed with Fe 3 O 4 magnetic nanoparticles); mixing vascular endothelial cells with collagen solution and loading the second nozzle assembly of the 3D printing device Mixing B lymphocytes with a collagen solution and loading it into a third nozzle assembly of the 3D printing device;
  • d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of the main structure of adipose stem cells / gelatin and hepatocytes / gelatin / fibrinogen, and use thrombin to polymerize fibrinogen; synchronous or asynchronous control of vascular endothelium Cell/collagen channel formation, obtaining two vascular channels; synchronous or asynchronous control of B lymphocyte/collagen channel formation, obtaining immune system channels; using glutaraldehyde solution to crosslink collagen into hydrogel, layer by layer, The multi-channel, three-channel, liver-like structure.
  • Example 4 A pancreatic structure containing one-in-one-out dual-vessel and three-channel nervous system was prepared by a multi-core rotation combined mold method, and the bionic pancreatic tissue was trained in a composite electromagnetic field.
  • the islet ⁇ cells as a host cell, mixed with the configured fibrinogen solution, the mass concentration of the fibrinogen solution in the mixture is 5%;
  • DMEM cell culture medium
  • step islet ⁇ cell/fibrinogen mixture into the inner cavity of the top mold through the macropores, and simultaneously rotating the mold base and the top mold at a rotation speed of 30 r/min, and the mold base and the mold branch core are relatively stationary.
  • the perfused mixture forms a semi-spindle shape, and thrombin is used to convert fibrinogen into a hydrogel during rotation;
  • adipose stem cells/DMEM into one-in-one double-tube of a three-channel biomimetic structure precursor to form a vascular system channel; injecting Schwann cells/DMEM into another channel of the channel biomimetic structure precursor to form The nervous system channel; eventually, a three-channel pancreatic structure is obtained.
  • Example 5 A liver structure containing four channels of blood vessels, bile ducts, nerves and immune system was prepared by a multi-core rotating combination mold method, and the liver structure was trained in a composite electromagnetic field.
  • hepatocytes and adipose stem as host cells, mixed with a configured sodium alginate/gelatin solution, fibrinogen solution, wherein the mixture has a volume concentration of sodium alginate and fibrinogen of 5%; in the mixture Adding 3% by volume of DMSO;
  • endothelial cells a) using endothelial cells, Schwann cells, and neutrophils as channel cells; mixing endothelial cells and Schwann cells with cell culture medium (DMEM); mixing neutrophils with sodium alginate solution;
  • DMEM cell culture medium
  • adipose stem cell/heparin/EGF/PBS suspension into the first channel of a four-channel biomimetic structure precursor to form a vascular system channel; perfusion of Schwann cell PBS suspension into a four-channel biomimetic structure precursor In the second channel, the nervous system channel is formed; the neutrophil/PBS suspension is perfused into the third channel of the four-channel biomimetic structure precursor, and CaCl 2 is cross-linked to form the immune system channel; finally, Four-channel semi-spindle liver structure.
  • the two semi-spindle liver precursors are adhered together with a sodium alginate/gelatin/DMSO solution, and a layer of synthetic high molecular polyurethane solution is sprayed to form a spindle-shaped liver precursor;
  • a spindle-shaped liver precursor structure that is driven by a motor to control the rotation of the magnetic field generating system, the magnitude of the magnetic field, and the action time so that the magnetic induction line also passes;
  • Example 6 A four-channel heart structure containing blood vessels, nerves, and immune system was prepared using a multi-core rotating combination mold method, and the heart structure was trained in an electric field.
  • the cardiomyocytes as a host cell, mixed with the configured fibrinogen solution, the mass concentration of the fibrinogen solution in the mixture is 5%; adding a mass volume concentration of 3% dextrose to the mixture;
  • DMEM cell culture medium
  • the myocardial/fibrinogen/dextrose solution mixture is perfused through the macropores into the inner cavity of the top mold, while the mold base and the top mold are relatively rotated, the rotation speed is 30r/min, and the mold base and the mold branch core are Relatively static, the perfused mixture forms a semi-spindle shape, and thrombin is used to convert fibrinogen to hydrogel during rotation;
  • adipose stem cells/EGF/b-FGF/paclitaxel/DMEM perfusion of adipose stem cells/EGF/b-FGF/paclitaxel/DMEM into the first channel of a four-channel biomimetic structure precursor to form a vascular system channel; perfusion of adipose stem cells and smooth muscle cells/DMEM into a four-channel biomimetic structure
  • a vascular system channel is formed; the neuron/DMEM is perfused into the third channel of the four-channel biomimetic structure precursor to form a nervous system channel; before the lymphocyte/DMEM is perfused into the four-channel biomimetic structure
  • the lymphatic passage of the immune system is formed; finally, a four-channel pseudo-cardiac structure is obtained.
  • the electric field generating device of the electromagnetic training device will be energized, and the current is controlled by a direct current alternating current of 30 mA;
  • Example 7 An artificial skin structure containing multiple channels of blood vessels, nerves, and immune system was prepared using a multi-channel detachable combination mold method, and the skin structure was trained in an electromagnetic field.
  • the fibroblasts as a host cell, mixed with the configured sodium alginate solution, the mass concentration of the sodium alginate solution in the mixture is 5%;
  • fibroblasts adipose stem cells as host cells, neurons and lymphocytes as channel cells, keratinocytes, epithelial cells as composite cells, respectively mixed in cell culture medium (DMEM);
  • the electric field generating device of the electromagnetic training device will be energized, and the control current is AC 20 mA;
  • the adipose stem cells as a host cell, mixed with the configured sodium alginate solution, the mass concentration of the sodium alginate solution in the mixture is 1%;
  • endothelial cells a) using endothelial cells, Schwann cells, and neutrophils as channel cells; mixing endothelial cells and Schwann cells with cell culture medium (DMEM); mixing neutrophils with sodium alginate solution;
  • DMEM cell culture medium
  • adipose stem cell/sodium alginate/DMSO solution mixture is poured into the inner cavity of the top mold through the macropores, and the mold base and the top mold are relatively rotated at a rotation speed of 30 r/min, and the mold base is opposite to the mold core. Static, so that the perfused mixture forms a semi-spindle shape, and the sodium alginate is converted into a hydrogel by using CaCl 2 during the rotation;
  • the two semi-spindle breast precursors are adhered together with a fibrinogen/adipose stem cell solution, and a layer of synthetic high molecular polylactic acid and polyglycolic acid copolymer (PLGA) solution is sprayed to form a spindle-shaped breast precursor.
  • PLGA synthetic high molecular polylactic acid and polyglycolic acid copolymer
  • the electric field line passes through the spindle-shaped breast precursor structure at this time;
  • a spindle-shaped breast precursor structure that controls the rotation of the magnetic field generating system, the magnitude of the magnetic field, and the action time by the motor so that the magnetic induction line also passes;
  • Example 9 A lung lobe structure containing two channels of blood vessels, nerves, and immune system was prepared by a combined mold method, and the lung lobe structure was trained in a magnetic field.
  • Adipose stem cells and lung epithelial cells are used as host cells, and mixed with the prepared gelatin solution, the mass concentration of the gelatin solution is 10%, respectively, and the cell cryopreservative glycerin is added in a mass fraction of 3%, and then the mixture is added.
  • Injection To the corresponding position of the combined mold, then inject or infuse the 1% collagen solution onto the corresponding cell layer, freeze the cell gelatin and collagen layer at 37 ° C for 30 minutes, or convert the collagen into a gel using a glutaraldehyde solution;
  • vascular endothelial cells and B lymphocytes as channel cells (both of which have been mixed with Fe 3 O 4 magnetic nanoparticles); mixing vascular endothelial cells with fibrinogen solution and injecting into the two-in and two-out vascular system Mixing B lymphocytes with fibrinogen solution and injecting into a branching system; mixing Schwann cells with fibrinogen solution into another branching nervous system;
  • Fibrinogen was hydrogeled with 0.1% thrombin solution to obtain a four-channel lobes structure.
  • Example 10 A liver-like tumor structure containing a vascular and a nervous system dual channel was prepared by a multi-nozzle 3D printing method, and the liver-like tumor structure was subjected to pulsation culture training in an electric field.
  • liver cancer cells are used as host cells, and mixed with the configured sodium alginate solution, the sodium alginate hydrogel solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
  • vascular endothelial cells and neurons as channel cells; mixing vascular endothelial cells with sodium alginate solution into a second nozzle assembly of a 3D printing device; mixing neurons with cell culture fluid (DMEM), loading Into the third nozzle assembly of the 3D printing device;
  • DMEM cell culture fluid
  • Example 11 A multi-nozzle 3D printing method was used to prepare a pseudo-chondral structure containing three channels of blood vessels, nerves and immune system, and the pseudo-chondral structure was trained in an electric field.
  • the chondrocytes are used as host cells, mixed with the configured fibrinogen solution, and the fibrinogen solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
  • vascular endothelial cells, smooth muscle cells, neurons, and T lymphocytes as channel cells; mixing vascular endothelial cells and smooth muscle cells with sodium alginate solution, and loading them into a second nozzle assembly of the 3D printing device; Mixing with the sodium alginate solution, loading into the third nozzle assembly of the 3D printing device; mixing the T lymphocytes with the sodium alginate solution into the fourth nozzle assembly of the 3D printing device;
  • the electric field generating device of the electromagnetic training device will be energized, and the control current is 2 mA, alternating current;

Abstract

A bionic structure containing channels and an electromagnetic force training device and method therefor. A multi-function and multi-system three-dimensional bionic structure containing channels is in vitro trained by the electromagnetic force training device. The structural main body of the channel is in a state with two through holes in two ends, a state with two blind holes in two ends or a state with a blind hole in one end and a through hole in the other end, and channels are intersecting, parallel, collinear or bifacial with each other. The bionic structure in the present invention is trained or subjected to pulsation training in electric, magnetic or composite electromagnetic fields, and cells are layer-oriented arranged in the micro-fluid channel. The bionic structure comprises at least one kind of cells. At least one kind of cells is distributed in the outer wall, inner wall or hole of the channel. The main body (101) of the bionic structure is a mixture of cells and natural polymer hydrogel. The bionic structure is a substitution for a tissue or an organ of a human body, and can be used for providing a carrier for high-throughput drug screening, and providing the possibility for organ transplantation.

Description

一种含通道的仿生结构及其电磁力训练装置和方法Bionic structure containing channel and electromagnetic force training device and method thereof 技术领域Technical field
本发明属于组织器官制造、生物工程及生物医疗器械领域，涉及一种含通道的仿生结构及其制备方法。The invention belongs to the field of tissue organ manufacturing, bioengineering and biomedical devices, and relates to a bionic structure containing channels and a preparation method thereof.
背景技术Background technique
目前，器官移植技术始终面临着免疫排斥，供体短缺，器官分配，伦理道德等一系列问题。据统计，我国每年需要进行器官移植的患者超过150万，但供求比不足1:100[Langer R.Tissue Engineering,2007,13(1):1～2]。组织工程学(Tissue Engineering)和器官制造(Organ Manufacturing)利用工程和医学相结合的概念，从组织和器官层面为这一难题有了崭新的解决途径。At present, organ transplantation technology has always faced a series of problems such as immune rejection, donor shortage, organ distribution, and ethics. According to statistics, more than 1.5 million patients need organ transplants each year in China, but the supply-demand ratio is less than 1:100 [Langer R. Tissue Engineering, 2007, 13(1): 1-2]. Tissue Engineering and Organ Manufacturing use a combination of engineering and medicine to create a new solution to this problem from the organizational and organ levels.
人工组织或器官的仿生结构可用于植入体内修复组织缺损，替代器官功能；也可或作为一种体外装置，暂时替代器官功能[曹谊林等,临床外科杂志,2007,15(1):40-41]。目前，利用组织工程学方法构建的组织器官已经在临床上实现了应用，但是这些应用多局限在软骨、上皮等组织，这些组织结构简单，成分单一，尺寸受限。而在复杂器官的组织工程学制造中，人们面临的主要难题是，在构建功能组织和支架结构的同时，构建相应的多功能器官替代物，这些功能主要包括循环系统、神经系统和免疫系统。The biomimetic structure of artificial tissue or organ can be used to implant the body to repair tissue defects, replacing organ function; or as an extracorporeal device, temporarily replacing organ function [Cao Yilin et al., Journal of Clinical Surgery, 2007, 15(1): 40- 41]. At present, tissue organs constructed by tissue engineering methods have been clinically applied, but these applications are mostly confined to tissues such as cartilage and epithelium. These tissues are simple in structure, single in composition, and limited in size. In the tissue engineering of complex organs, the main problem is to construct functional multi-organ substitutes while constructing functional tissues and scaffold structures. These functions mainly include circulatory system, nervous system and immune system.
人体内的重要脏器如心脏、肾脏和肝脏等，都有着复杂的血管、神经、免疫和内分泌系统。细胞存活的适宜环境是在血管周围150～200μm的范围内，否则很快会因物质交流不畅导致死亡；而建立神经系统的联系是移植器官与人体交流的重要途径，这对复杂器官行使功能，参与人体调节系统都有着重大意义。综上，如何构多系统复合的含通道的仿生结构是复杂器官制造中不可避免的问题。Important organs in the human body, such as the heart, kidneys and liver, have complex vascular, neurological, immune and endocrine systems. The suitable environment for cell survival is in the range of 150-200 μm around the blood vessels, otherwise it will soon die due to poor material communication; and establishing the connection of the nervous system is an important way for the transplanted organs to communicate with the human body, which functions for complex organs. Participation in the human body regulation system has great significance. In summary, how to construct a multi-system composite channel-containing bionic structure is an inevitable problem in the manufacture of complex organs.
另外，目前的含通道的组织/器官仿生结构虽有管道结构来模拟体内的复杂导管，但是这些管道很难在微观层面形成可控的单层或多层细胞层状结构，这要归咎于目前成形技术在微观尺度上的局限性。因此，我们希望采用外加场的作用，在微观尺度上控制微流体管道中单层细胞的附着和定向排列。本发明采用外加电场、磁场或复合电磁场的作用，对带电粒子(主要是溶液粒子、普通细胞、磁性细胞和带电细胞)进行排列控制，使得细胞能够按照预设层数排布在管道壁上，得到更类似体内导管的细胞排列样式，达到对细胞的精细操作。In addition, current channel-containing tissue/organ bionic structures have a pipeline structure to simulate complex conduits in vivo, but these pipelines are difficult to form a controlled monolayer or multi-layer cell layer structure at the microscopic level, which is due to the current The limitations of forming techniques on a micro scale. Therefore, we hope to control the attachment and orientation of monolayer cells in microfluidic channels on a microscopic scale using the role of an external field. The invention adopts the action of an external electric field, a magnetic field or a composite electromagnetic field to arrange and control the charged particles (mainly solution particles, ordinary cells, magnetic cells and charged cells), so that the cells can be arranged on the pipeline wall according to the preset number of layers. A cell arrangement pattern that is more similar to a catheter in the body is obtained to achieve fine manipulation of the cells.
清华大学器官制造中心(Center of Organ Manufacturing)采用3D打印技术和旋转组合模具法成功制备出具有一定功能的组织或器官前体。利用现有的单/双喷头快速成形技术(也称3D打印技术)，该中心已成功制备出简单的血管网、肝组织和骨修复材料等[Wang X,et al.Trends in Biotechnology,2007,25:505；Wang X.Artificial organs,2012,36:591]。利用旋转组合模具法[专利201210324600.4]，该中心已设计出具有通道的血管化器官前体。但是，这些技术目前能制 备的结构局限在简单的组织，仅含类血管的单一通道，并未应用于多系统(血管、神经和免疫)复合的组织/器官。另外，现有办法仅能制备出一般管状结构，尚不能在管状结构孔内或管壁上灌注或定位细胞。因此我们提出利用多喷头3D打印法和多内芯旋转组合模具法为基础，采用外加场的作用，在微观尺度上控制微流体管道中单层细胞的附着和定向排列，来制备更为贴近人体组织或器官的仿生结构，该仿生结构复合了循环系统、神经系统、免疫系统和内分泌系统等功能，将复杂器官制造技术进一步推进。The Center of Organ Manufacturing of Tsinghua University successfully produced a certain function of tissue or organ precursors using 3D printing technology and rotating combination mold method. Using existing single/double nozzle rapid prototyping technology (also known as 3D printing technology), the center has successfully produced simple vascular networks, liver tissue and bone repair materials [Wang X, et al. Trends in Biotechnology, 2007, 25:505; Wang X. Artificial organs, 2012, 36: 591]. Using the rotary combined mold method [Patent 201210324600.4], the center has designed a vascularized organ precursor with a channel. However, these technologies are currently available The structure of the preparation is limited to a simple tissue, containing only a single channel of blood vessels, and is not applied to multiple systems (vascular, neurological, and immune) composite tissues/organs. In addition, the existing method can only produce a general tubular structure, and it is not possible to perfuse or locate cells in the tubular structure pores or on the tube wall. Therefore, we propose to use the multi-nozzle 3D printing method and the multi-core rotating combined mold method to control the attachment and orientation of single-layer cells in the microfluidic pipeline on the microscopic scale, based on the action of the external field. The biomimetic structure of a tissue or organ that combines the functions of the circulatory system, the nervous system, the immune system, and the endocrine system to further advance the manufacturing technology of complex organs.
通过以上分析，现有技术条件下的组织器官替代物形貌和功能单一，往往仅具有一种欠成熟的循环体统(血管网络)，在复杂器官制造和功能发挥方面尚不完善。将3D打印法或旋转组合模具法与器官制造技术结合是医学和工程学的研究热点。Through the above analysis, the morphology and function of the tissue and organ substitutes under the prior art conditions are single, and often only have an underdeveloped circulation system (vascular network), which is still not perfect in the manufacture and function of complex organs. Combining 3D printing or rotating combination mold methods with organ manufacturing techniques is a research hotspot in medicine and engineering.
发明内容Summary of the invention
本发明的目的是提供一种含通道的仿生结构，该结构具备含血管、神经和免洗系统的细胞管道，使其具有真实器官的结构和功能。本发明的另一目的是提供一种电磁力训练装置及其训练通道仿生结构的方法，使细胞在电场、磁场或复合电磁场中完成逐层定位，更贴近生物体内部管道的细胞排列。It is an object of the present invention to provide a channel-containing biomimetic structure having a cell conduit containing blood vessels, nerves, and an abrad system that has the structure and function of a real organ. Another object of the present invention is to provide an electromagnetic force training device and a method for training the biomimetic structure thereof, so that the cells are positioned layer by layer in an electric field, a magnetic field or a composite electromagnetic field, and are closer to the cell arrangement of the internal conduit of the living body.
本发明的技术方案如下：The technical solution of the present invention is as follows:
一种含通道的仿生结构，其特征在于：所述仿生结构包括结构主体和至少一个通道，所述通道分布在结构主体内；所述结构主体为天然高分子水凝胶和细胞组成的混合物，所述细胞浓度为10-108个/mL，该细胞为胚胎干细胞、成体干细胞、成体细胞、癌细胞和诱导多能干细胞中的至少一种；所述通道为单通道或分支通道，通道为两端通孔、两端盲孔和一端盲孔一端通孔结构中的一种或几种组合；通道之间的位置关系为相交、平行、共线或异面；所述通道外壁、通道内壁或通道孔内分布有细胞，细胞为神经系统种子细胞、血管系统种子细胞和免疫系统种子细胞中的至少两种。A channel-containing biomimetic structure, characterized in that the biomimetic structure comprises a structural body and at least one channel, the channel is distributed in the structural body; the structural body is a mixture of a natural polymer hydrogel and a cell. The cell concentration is 10-10 8 /mL, and the cell is at least one of an embryonic stem cell, an adult stem cell, an adult cell, a cancer cell, and an induced pluripotent stem cell; the channel is a single channel or a branch channel, and the channel is One or more combinations of through-holes at both ends, blind holes at both ends, and one-end through-hole structures at one end; the positional relationship between the channels is intersecting, parallel, collinear or different; the outer wall of the channel and the inner wall of the channel Or cells are distributed in the pores of the channel, and the cells are at least two of the seed cells of the nervous system, the seed cells of the vascular system, and the seed cells of the immune system.
上述技术方案中，所述所述通道的截面为圆形、椭圆形、多边形或不规则几何图形，且通道截面面积为100μm2-1cm2。所述的一种含通道的仿生结构，其特征在于：所述神经系统种子细胞为神经元和神经胶质细胞中的至少一种；所述血管种子细胞为内皮细胞、平滑肌细胞和脂肪干细胞中的至少一种；所述免疫系统种子细胞为淋巴细胞和固有免疫细胞中的至少一种。In the above technical solution, the cross section of the channel is circular, elliptical, polygonal or irregular geometrical, and the channel cross-sectional area is 100 μm 2 -1 cm 2 . The channel-containing biomimetic structure is characterized in that: the nervous system seed cells are at least one of neurons and glial cells; and the blood vessel seed cells are endothelial cells, smooth muscle cells and adipose stem cells. At least one of the immune system seed cells is at least one of lymphocytes and innate immune cells.
本发明所述天然高分子水凝胶为海藻酸钠、胶原、基质胶、右旋糖、壳聚糖、明胶和纤维蛋白原中的至少一种，该水凝胶的质量体积浓度为0.1～20％。所述天然高分子水凝胶中复合有细胞冻存剂、细胞生长因子、药物、抗凝血剂和磁性纳米颗粒中的至少一种；所述细胞冻存剂为二甲基亚砜、甘油和右旋糖中的至少一种；所述细胞生长因子为血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子、人血小板衍化生长因子和转化生长因子中的至少一种；所述的药物为抗肿瘤药物和病毒疫苗中的至少一种；所述的抗凝血剂为肝素和紫杉醇中的至少一种；所述磁性纳米颗粒为铁氧体颗粒、金属型颗粒和氮化铁颗粒中的至少一种。 The natural polymer hydrogel of the present invention is at least one of sodium alginate, collagen, matrigel, dextrose, chitosan, gelatin and fibrinogen, and the mass concentration of the hydrogel is 0.1 to 20%. The natural polymer hydrogel is compounded with at least one of a cell cryopreservative, a cell growth factor, a drug, an anticoagulant, and a magnetic nanoparticle; the cell cryopreservative is dimethyl sulfoxide, glycerin And at least one of dextrose; the cell growth factor is at least one of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, human platelet derived growth factor, and transforming growth factor; The drug is at least one of an antitumor drug and a virus vaccine; the anticoagulant is at least one of heparin and paclitaxel; the magnetic nanoparticles are ferrite particles, metal particles and nitriding At least one of the iron particles.
本发明提供的一种含通道的仿生结构的电磁力训练装置，其特征在于：所述装置包括电场发生系统、磁场发生系统、样品台和固定平台；所述磁场发生系统和样品台安装于固定平台上；所述电场发生系统含正极和负极；所述样品台设有导轨，所述正极和负极分别通过滑块安装在导轨上；所述样品台位于磁场发生系统内；所述磁场发生系统分包括支架和可旋转圆环，所述可旋转圆环安装在支架的中空结构内，在可旋转圆环上径向对称排布有可拆卸的S极和N极。The present invention provides an electromagnetic force training device for a channel-containing bionic structure, characterized in that the device comprises an electric field generating system, a magnetic field generating system, a sample stage and a fixed platform; the magnetic field generating system and the sample stage are fixed at a fixed position On the platform; the electric field generating system includes a positive electrode and a negative electrode; the sample stage is provided with a guide rail, and the positive electrode and the negative electrode are respectively mounted on the guide rail through a slider; the sample stage is located in the magnetic field generating system; the magnetic field generating system The sub-assembly includes a bracket and a rotatable ring mounted in the hollow structure of the bracket, and a detachable S pole and N pole are radially symmetrically arranged on the rotatable ring.
本发明所述的装置中还可包括脉动培养系统；所述脉动培养系统安装于固定平台上；所述脉动培养系统包括脉动系统电机、脉动系统导轨-滑块机构、培养液供给注射器、单向阀、导流管和培养液瓶；所述脉动系统电机通过曲柄与脉动系统导轨-滑块机构连接；所述脉动系统导轨-滑块机构与培养液供给注射器连接。The device of the present invention may further comprise a pulsation culture system; the pulsation culture system is mounted on a fixed platform; the pulsation culture system comprises a pulsation system motor, a pulsation system guide rail-slider mechanism, a culture fluid supply injector, and a unidirectional a valve, a draft tube and a culture fluid bottle; the pulsation system motor is coupled to the pulsation system rail-slider mechanism by a crank; the pulsation system rail-slider mechanism is coupled to the culture fluid supply injector.
本发明所述的装置中，所述正极和负极为金属平板、金属导线或金属探针中的一种。所述S极和N极为永磁体或电磁铁中的一种。In the device of the present invention, the positive electrode and the negative electrode are one of a metal plate, a metal wire or a metal probe. The S pole and the N are one of a permanent magnet or an electromagnet.
本发明还提供了一种利用上述装置的电场来训练仿生结构的方法，其特征在于，所述方法包括如下步骤：The present invention also provides a method of training a biomimetic structure using an electric field of the above apparatus, characterized in that the method comprises the following steps:
a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
b)启动电磁训练装置的电场发生系统，移除磁场发生系统的N级和S级；使含通道的仿生结构位于电场中；电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；b) starting the electric field generating system of the electromagnetic training device, removing the N and S stages of the magnetic field generating system; and making the bionic structure containing the channel in the electric field; the current is greater than 0, less than or equal to 50 mA; the voltage is greater than 0, less than or equal to 50 V The current direction is alternating between AC, DC or both;
c)完成电场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the training of the electric field on the bionic structure containing the channel, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
本发明还提供了一种利用上述装置的磁场来训练仿生结构的方法，其特征在于，所述方法包括如下步骤：The present invention also provides a method of training a biomimetic structure using the magnetic field of the above apparatus, characterized in that the method comprises the following steps:
a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
b)关闭电磁训练装置的电场发生系统，启动磁场发生系统，使S极和N极随可旋转圆环旋转，控制磁感应强度为大于0，小于等于5T；b) turning off the electric field generating system of the electromagnetic training device, starting the magnetic field generating system, so that the S pole and the N pole rotate with the rotatable ring, and the control magnetic induction intensity is greater than 0, less than or equal to 5T;
c)完成磁场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the training of the magnetic field on the channel-containing bionic structure, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
本发明还提供了一种利用上述装置的电磁复合场来训练仿生结构的方法，其特征在于，所述方法包括如下步骤：The present invention also provides a method of training a biomimetic structure using an electromagnetic composite field of the above apparatus, characterized in that the method comprises the following steps:
a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上； a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
b)同时启动电磁训练装置的电场发生系统和磁场发生系统，使含通道的仿生结构位于电场和磁场中；控制电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；控制磁感应强度为大于0，小于等于5T；b) simultaneously start the electric field generating system and the magnetic field generating system of the electromagnetic training device, so that the bionic structure containing the channel is located in the electric field and the magnetic field; the control current is greater than 0, less than or equal to 50 mA; the voltage is greater than 0, less than or equal to 50 V; AC, DC or both are used alternately; the control magnetic induction intensity is greater than 0, less than or equal to 5T;
c)完成电场和磁场同时对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the electric field and magnetic field training of the channel-containing bionic structure, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
在上述电场、磁场和电磁复合场训练仿生结构的方法的基础上，本发明提供了将脉动培养系统同时应用其中的训练方法，其特征在于：启动脉动培养系统，所述脉动培养系统包括脉动系统电机、脉动系统导轨-滑块机构、培养液供给注射器、单向阀、导流管和培养液瓶；所述脉动系统电机通过曲柄与脉动系统导轨-滑块机构连接；所述导轨-滑块机构与培养液供给注射器连接；通过导流管将所述仿生结构与脉动培养系统连接；使培养液在导流管和含通道的仿生结构之间进行单向流动；液体流速为大于0，小于30cm/s，进行脉动-电场、脉动-磁场或脉动-电磁复合场的作用；完成对所述仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。Based on the above method for training a bionic structure by an electric field, a magnetic field and an electromagnetic composite field, the present invention provides a training method in which a pulsation culture system is simultaneously applied, characterized in that a pulsation culture system is started, and the pulsation culture system includes a pulsation system. a motor, a pulsating system guide rail-slider mechanism, a culture fluid supply injector, a check valve, a draft tube, and a culture fluid bottle; the pulsation system motor is connected to the pulsation system rail-slider mechanism by a crank; the guide rail-slider The mechanism is connected with the culture liquid supply syringe; the bionic structure is connected to the pulsation culture system through the flow guiding tube; the culture liquid is unidirectionally flowed between the draft tube and the bionic structure containing the channel; the liquid flow rate is greater than 0, less than At 30 cm/s, the action of the pulsation-electric field, the pulsation-magnetic field or the pulsation-electromagnetic composite field is performed; after the training of the bionic structure is completed, the bionic structure is cryopreserved, or the in vitro culture is continued or directly used for organ transplantation.
本发明与现有技术相比，有以下优点及突出性的技术效果：Compared with the prior art, the present invention has the following advantages and outstanding technical effects:
①本发明以现有的三维成形技术如3D打印法和旋转组合模具法为基础，将细胞和高分子凝胶混合物进一步复合，制备通道、多系统、多功能复杂组织器仿生结构。该含通道的仿生结构囊括了血管系统、神经系统和免疫系统，在结构上和功能上极大程度地模拟了体内真实的多系统的器官状态。1 The invention is based on the existing three-dimensional forming technology such as 3D printing method and rotating combined mold method, and further compounding the cell and polymer gel mixture to prepare a bionic structure of channel, multi-system and multifunctional complex organizer. The channel-containing biomimetic structure encompasses the vascular system, the nervous system, and the immune system, and structurally and functionally mimics the true multi-system state of the body.
②本发明的一种电磁力训练装置，结合了电场、磁场和脉动培养等作用，对仿生结构进行体外训练和力学培养，能使细胞在复合场的作用下逐层定位、排布于管道结构周边，贴近真实器官导管的细胞形貌，所成形的结构在形态结构上、免疫生理方面都有良好的性能。2 The electromagnetic force training device of the invention combines the functions of electric field, magnetic field and pulsation culture, and performs in vitro training and mechanical culture on the bionic structure, so that the cells can be positioned layer by layer and arranged in the pipeline structure under the action of the composite field. Peripheral, close to the cell morphology of the real organ duct, the formed structure has good performance in morphology, immunophysiology.
③本发明的一种电磁力训练装置的使用方法，能单独使用电场、单独使用磁场或将电磁场同时符合使用；此外，脉动培养系统的引进也增加了装置功能的多元化。3 The use of an electromagnetic force training device according to the present invention can use an electric field alone, use a magnetic field alone or simultaneously use an electromagnetic field; in addition, the introduction of a pulsation culture system also increases the diversification of device functions.
附图说明DRAWINGS
图1为含通道的仿生组织结构示意图。Figure 1 is a schematic diagram of a bionic tissue structure containing channels.
图2a～2i为几种通道细胞的状态示意图；其中，图2a为一种细胞位于通道外壁的示意图，图2b为一种细胞位于通道内壁的示意图，图2c为一种细胞位于通道孔内的示意图，图2d为两种细胞位于通道外壁的示意图，图2e为两种细胞位于通道内壁的示意图，图2f为两种细胞位于通道孔内的示意图，图2g为一种细胞位于通道外壁另一种细胞位于通道孔内的示意图，图2h为一种细胞位于通道内壁另一种细胞位于通道孔内的示意图，图2i为一种细胞位于通道外壁另一种细胞位于通道内壁的示意图。2a-2i are schematic views of the state of several channel cells; wherein, FIG. 2a is a schematic diagram of a cell located on the outer wall of the channel, FIG. 2b is a schematic view of a cell located on the inner wall of the channel, and FIG. 2c is a view of a cell located in the channel hole. Schematic diagram, Fig. 2d is a schematic diagram of two cells located on the outer wall of the channel, Fig. 2e is a schematic diagram of two cells located on the inner wall of the channel, Fig. 2f is a schematic diagram of two cells in the channel hole, and Fig. 2g is a cell located on the outer wall of the channel. 2h is a schematic diagram of a cell located in the channel wall and another cell located in the channel hole. FIG. 2i is a schematic diagram of a cell located on the outer wall of the channel and another cell located on the inner wall of the channel.
图3为采用多喷头3D打印法成形含通道的仿生结构示意图。Fig. 3 is a schematic view showing the formation of a bionic structure containing a channel by a multi-nozzle 3D printing method.
图4a和图4b分别为多内芯旋转组合模具法成形通道仿生结构的示意图和爆炸图。 4a and 4b are respectively a schematic view and an exploded view of a bionic structure of a multi-core rotating combined mold forming channel.
图5a、5b和5c分别为纺锤状双通道仿生结构示意图、块状交叉通道仿生结构示意图和块状平行通道仿生结构示意图。5a, 5b and 5c are respectively a schematic view of a spindle-shaped two-channel biomimetic structure, a schematic diagram of a block-shaped cross-channel bionic structure and a block-like parallel channel bionic structure.
图6为电磁训练装置示意图。Figure 6 is a schematic view of an electromagnetic training device.
图7a为磁场发生系统爆炸示意图，图7b为脉动培养系统示意图。Figure 7a is a schematic diagram of the explosion of the magnetic field generating system, and Figure 7b is a schematic diagram of the pulsating culture system.
图8a为含通道的仿生结构位于金属平板电极之间，图8b为通道的仿生结构位于金属导线或金属探针之间，图8c为带电粒子在电场(E)中的运动示意图。Figure 8a shows the bionic structure containing the channel between the metal plate electrodes, Figure 8b shows the bionic structure of the channel between the metal wire or the metal probe, and Figure 8c is a schematic diagram of the movement of the charged particles in the electric field (E).
图9a为含通道的仿生结构位于磁场S、N极之间，图9b为磁性粒子在磁场(B)中的运动示意图(虚线表示磁场转动方向)。Fig. 9a shows that the bionic structure containing the channel is located between the magnetic field S and the N pole, and Fig. 9b is a schematic view of the movement of the magnetic particle in the magnetic field (B) (the broken line indicates the direction of the magnetic field rotation).
图10a为含通道的仿生结构位于电场正、负极和磁场S、N极之间，图10b为带电粒子在复合电磁场中的运动示意图(虚线表示磁场转动方向)。Fig. 10a shows that the bionic structure containing the channel is located between the positive and negative electric fields and the magnetic field S and N, and Fig. 10b is a schematic diagram of the movement of the charged particles in the composite electromagnetic field (the broken line indicates the direction of rotation of the magnetic field).
图中：101-结构主体；102-位于主体的细胞；103-两端通孔的通道；104-一端盲孔一端通孔的通道；105-两端盲孔的通道；106-含分支通道；107-位于通道孔内的细胞；108-位于通道外壁的细胞；109-位于通道内壁的细胞；301-3D打印喷头组件；302-3D打印的结构主体；303-3D打印的圆形截面的通道；304-3D打印的不规则截面的通道；401-组合模具底座；402-组合模具分支内芯；403-组合模具上模具；404-组合模具底座插孔；501-含多通道的仿生结构主体；502-含多通道的仿生结构的通道；601-电场发生系统(正极和负极)；602-支架；603-样品台；604-电机驱动；605-固定平台；606-N极；607-S极；608-可旋转圆环；701-脉动培养系统；702-脉动系统电机；703-脉动系统导轨-滑块机构；704-培养液供给注射器；705-单向阀；706-导流管；707-纺锤状通道仿生结构；708-培养液瓶。In the figure: 101 - structural body; 102 - cells located in the body; 103 - channels through the through holes at both ends; 104 - channels through which one end of the blind holes is open; 105 - channels with blind holes at both ends; 106 - branch channels; 107- cells located in the channel hole; 108- cells located on the outer wall of the channel; 109- cells located on the inner wall of the channel; 301-3D print head assembly; 302-3D printed structure body; 303-3D printed circular section channel ;304-3D printed irregular section channel; 401-combined mold base; 402-combined mold branch inner core; 403-combined mold upper mold; 404-combined mold base socket; 501-multi-channel bionic structural body 502-channel with multi-channel bionic structure; 601- electric field generating system (positive and negative); 602-bracket; 603-sample stage; 604-motor drive; 605-fixed platform; 606-N pole; 607-S Pole; 608-rotatable ring; 701-pulsation culture system; 702-pulsation system motor; 703-pulsation system guide-slider mechanism; 704-culture fluid supply injector; 705-check valve; 706-drainage tube; 707-spindle channel biomimetic structure; 708-culture flask.
具体实施方式detailed description
下面结合附图和实施例对本发明进一步说明。The invention will now be further described with reference to the drawings and embodiments.
如图1所示，一种含通道的仿生结构，包括结构主体101和至少一个通道，所述通道分布在结构主体101内；所述结构主体为天然高分子水凝胶和细胞组成的混合物，所述细胞浓度为10-108个/mL，该细胞为胚胎干细胞、成体干细胞、成体细胞、癌细胞和诱导多能干细胞中的至少一种；所述通道为单通道或分支通道105，通道为两端通孔103、两端盲孔106和一端盲孔一端通孔结构104中的一种或几种组合；通道之间的位置关系为相交、平行、共线或异面；所述通道外壁、通道内壁或通道孔内分布有细胞，细胞为神经系统种子细胞、血管系统种子细胞和免疫系统种子细胞中的至少两种。所述通道的截面为圆形、椭圆形、多边形或不规则几何图形，且通道截面面积为100μm2-1cm2。所述神经系统种子细胞为神经元和神经胶质细胞中的至少一种；所述血管种子细胞为内皮细胞、平滑肌细胞和脂肪干细胞中的至少一种；所述免疫系统种子细胞为淋巴细胞和固有免疫细胞中的至少一种。As shown in FIG. 1, a channel-containing biomimetic structure includes a structural body 101 and at least one channel distributed in the structural body 101; the structural body is a mixture of a natural polymer hydrogel and a cell. The cell concentration is 10-10 8 /mL, the cell is at least one of an embryonic stem cell, an adult stem cell, an adult cell, a cancer cell and an induced pluripotent stem cell; the channel is a single channel or a branch channel 105, the channel One or more combinations of the through holes 103 at both ends, the blind holes 106 at both ends, and the one end through holes 104 at one end; the positional relationship between the channels is intersecting, parallel, collinear or different; Cells are distributed in the outer wall, the inner wall of the channel or the pores of the channel, and the cells are at least two of the seed cells of the nervous system, the seed cells of the vascular system, and the seed cells of the immune system. The channel has a circular, elliptical, polygonal or irregular geometry and has a channel cross-sectional area of 100 μm 2 -1 cm 2 . The nervous system seed cell is at least one of a neuron and a glial cell; the blood vessel seed cell is at least one of an endothelial cell, a smooth muscle cell, and an adipose stem cell; the immune system seed cell is a lymphocyte and At least one of innate immune cells.
图2a～2i为几种通道细胞的状态示意图；其中，图2a为一种细胞位于通道外壁的示意图，图2b为一种细胞位于通道内壁的示意图，图2c为一种细胞位于通道孔内的示意图，图2d为两种细胞位于通道外壁的示意图，图2e为两种细胞位于通道内壁的示意图，图2f为两种细 胞位于通道孔内的示意图，图2g为一种细胞位于通道外壁另一种细胞位于通道孔内的示意图，图2h为一种细胞位于通道内壁另一种细胞位于通道孔内的示意图，图2i为一种细胞位于通道外壁另一种细胞位于通道内壁的示意图。如图3、图4a和图4b所示，为仿生结构的制备方法。图3为采用多喷头3D打印法成形含通道的仿生结构示意图。图4a和图4b分别为多内芯旋转组合模具法成形通道仿生结构的示意图和爆炸图。2a-2i are schematic views of the state of several channel cells; wherein, FIG. 2a is a schematic diagram of a cell located on the outer wall of the channel, FIG. 2b is a schematic view of a cell located on the inner wall of the channel, and FIG. 2c is a view of a cell located in the channel hole. Schematic diagram, Fig. 2d is a schematic diagram of two cells located on the outer wall of the channel, Fig. 2e is a schematic diagram of two cells located on the inner wall of the channel, and Fig. 2f is two thin Schematic diagram of the cell located in the pore of the channel, FIG. 2g is a schematic diagram of another cell located in the channel pore on the outer wall of the channel, and FIG. 2h is a schematic diagram of another cell located in the channel hole on the inner wall of the channel, FIG. 2i A schematic diagram of a cell located on the outer wall of the channel and another cell located on the inner wall of the channel. As shown in FIG. 3, FIG. 4a and FIG. 4b, it is a preparation method of a biomimetic structure. Fig. 3 is a schematic view showing the formation of a bionic structure containing a channel by a multi-nozzle 3D printing method. 4a and 4b are respectively a schematic view and an exploded view of a bionic structure of a multi-core rotating combined mold forming channel.
图5a～5c为几种含多通道的仿生结构示意图。图5a、5b和5c分别为纺锤状双通道仿生结构示意图、块状交叉通道仿生结构示意图和块状平行通道仿生结构示意图。所述天然高分子水凝胶为海藻酸钠、胶原、基质胶、右旋糖、壳聚糖、明胶和纤维蛋白原中的至少一种，该水凝胶的质量体积浓度为0.1～20％。所述天然高分子水凝胶中复合有细胞冻存剂、细胞生长因子、药物、抗凝血剂和磁性纳米颗粒中的至少一种；所述细胞冻存剂为二甲基亚砜、甘油和右旋糖中的至少一种；所述细胞生长因子为血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子、人血小板衍化生长因子和转化生长因子中的至少一种；所述的药物为抗肿瘤药物和病毒疫苗中的至少一种；所述的抗凝血剂为肝素和紫杉醇中的至少一种；所述磁性纳米颗粒为铁氧体颗粒、金属型颗粒和氮化铁颗粒中的至少一种。Figures 5a to 5c are schematic views of several biomimetic structures containing multiple channels. 5a, 5b and 5c are respectively a schematic view of a spindle-shaped two-channel biomimetic structure, a schematic diagram of a block-shaped cross-channel bionic structure and a block-like parallel channel bionic structure. The natural polymer hydrogel is at least one of sodium alginate, collagen, matrigel, dextrose, chitosan, gelatin and fibrinogen, and the mass concentration of the hydrogel is 0.1 to 20%. . The natural polymer hydrogel is compounded with at least one of a cell cryopreservative, a cell growth factor, a drug, an anticoagulant, and a magnetic nanoparticle; the cell cryopreservative is dimethyl sulfoxide, glycerin And at least one of dextrose; the cell growth factor is at least one of vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, human platelet derived growth factor, and transforming growth factor; The drug is at least one of an antitumor drug and a virus vaccine; the anticoagulant is at least one of heparin and paclitaxel; the magnetic nanoparticles are ferrite particles, metal particles and nitriding At least one of the iron particles.
如图6所示，一种电磁训练装置，包括电场发生系统601、磁场发生系统、样品台603和固定平台605；所述磁场发生系统和样品台603安装于固定平台605上；所述电场发生系601统含正极和负极；所述样品台603设有导轨，所述正极和负极分别通过滑块安装在导轨上；所述样品台603位于磁场发生系统内；所述磁场发生系统分包括支架602和可旋转圆环608，所述可旋转圆环608安装在支架602的中空结构内，在可旋转圆环608上径向对称排布有可拆卸的S极606和N极607，详见图7a。所述装置还可包括脉动培养系统701；所述脉动培养系统安装于固定平台605上；所述脉动培养系统701包括脉动系统电机702、脉动系统导轨-滑块机构703、培养液供给注射器704、单向阀705、导流管706和培养液瓶708；所述脉动系统电机702通过曲柄与脉动系统导轨-滑块机构703连接；所述脉动系统导轨-滑块机构703与培养液供给注射器704连接，详见图7b。所述正极和负极为金属平板、金属导线或金属探针中的一种。所述S极和N极为永磁体或电磁铁中的一种。As shown in FIG. 6, an electromagnetic training device includes an electric field generating system 601, a magnetic field generating system, a sample stage 603, and a fixed platform 605; the magnetic field generating system and sample stage 603 are mounted on a fixed platform 605; The system 601 includes a positive electrode and a negative electrode; the sample stage 603 is provided with a guide rail, and the positive electrode and the negative electrode are respectively mounted on the guide rail through a slider; the sample stage 603 is located in the magnetic field generating system; and the magnetic field generating system includes a bracket 602 and a rotatable ring 608, the rotatable ring 608 is mounted in the hollow structure of the bracket 602, and the detachable S pole 606 and the N pole 607 are radially symmetrically arranged on the rotatable ring 608. Figure 7a. The device may further include a pulsation culture system 701; the pulsation culture system is mounted on the fixed platform 605; the pulsation culture system 701 includes a pulsation system motor 702, a pulsation system guide-slider mechanism 703, a culture fluid supply injector 704, a check valve 705, a draft tube 706 and a culture fluid bottle 708; the pulsation system motor 702 is connected to the pulsation system rail-slider mechanism 703 by a crank; the pulsation system rail-slider mechanism 703 and the culture fluid supply syringe 704 Connection, see Figure 7b. The positive electrode and the negative electrode are one of a metal plate, a metal wire or a metal probe. The S pole and the N are one of a permanent magnet or an electromagnet.
如图8a所示，含通道的仿生结构位于金属平板电极之间，图8b为含通道的仿生结构位于金属导线或金属探针之间，图8c为带电粒子在电场(E)中的运动示意图。本发明将对含通道的仿生结构置于所述装置电场中进行训练的方法包括如下步骤：a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；b)启动电磁训练装置的电场发生系统，移除磁场发生系统的N级和S级；使含通道的仿生结构位于电场中；电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；c)完成电场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。 As shown in FIG. 8a, the channel-containing biomimetic structure is located between the metal plate electrodes, and FIG. 8b shows that the channel-containing bionic structure is located between the metal wires or the metal probes, and FIG. 8c is a schematic diagram of the movement of the charged particles in the electric field (E). . The method for training a channel-containing biomimetic structure in an electric field of the device includes the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture liquid containing suspended cells, and placing the electromagnetic force On the sample stage of the training device; b) starting the electric field generating system of the electromagnetic training device, removing the N and S stages of the magnetic field generating system; and making the bionic structure containing the channel in the electric field; the current is greater than 0, less than or equal to 50 mA; It is greater than 0, less than or equal to 50V; the current direction is alternately used by alternating current, direct current or both; c) after completing the training of the electric field on the bionic structure containing the channel, the biomimetic structure is cryopreserved or continuously cultured in vitro or directly used Organ transplantation.
如图9a所示，含通道的仿生结构位于磁场S、N极之间，图9b为磁性粒子在磁场(B)中的运动示意图(虚线表示磁场转动方向)。本发明将对含通道的仿生结构置于所述装置磁场中进行训练的方法包括如下步骤：a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；b)关闭电磁训练装置的电场发生系统，启动磁场发生系统，使S极和N极随可旋转圆环旋转，控制磁感应强度为大于0，小于等于5T；c)完成磁场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。As shown in Fig. 9a, the channel-containing biomimetic structure is located between the magnetic field S and the N pole, and Fig. 9b is a schematic diagram of the movement of the magnetic particles in the magnetic field (B) (the broken line indicates the direction of the magnetic field rotation). The method for training a channel-containing biomimetic structure in a magnetic field of the device includes the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture liquid containing suspended cells, and placing the electromagnetic force On the sample stage of the training device; b) turning off the electric field generating system of the electromagnetic training device, starting the magnetic field generating system, rotating the S pole and the N pole with the rotatable ring, controlling the magnetic induction intensity to be greater than 0, less than or equal to 5T; c) completing After the magnetic field is trained on the bionic structure containing the channel, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
图10a为含通道的仿生结构位于电场正、负极和磁场S、N极之间，图10b为带电粒子在复合电磁场中的运动示意图(虚线表示磁场转动方向)。本发明将对含通道的仿生结构置于所述装置电磁复合场中进行训练的方法包括如下步骤：a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；b)同时启动电磁训练装置的电场发生系统和磁场发生系统，使含通道的仿生结构位于电场和磁场中；控制电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；控制磁感应强度为大于0，小于等于5T；c)完成电场和磁场同时对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。Fig. 10a shows that the bionic structure containing the channel is located between the positive and negative electric fields and the magnetic field S and N, and Fig. 10b is a schematic diagram of the movement of the charged particles in the composite electromagnetic field (the broken line indicates the direction of rotation of the magnetic field). The method for training a channel-containing biomimetic structure in the electromagnetic composite field of the device comprises the following steps: a) immersing the prepared channel-containing biomimetic structure in a cell culture fluid containing suspended cells, and placing On the sample stage of the electromagnetic force training device; b) simultaneously starting the electric field generating system and the magnetic field generating system of the electromagnetic training device, so that the bionic structure containing the channel is located in the electric field and the magnetic field; the control current is greater than 0, less than or equal to 50 mA; and the voltage is greater than 0, less than or equal to 50V; current direction is alternating between alternating current, direct current or both; controlling magnetic induction intensity is greater than 0, less than or equal to 5T; c) after completing the electric field and magnetic field simultaneously training the bionic structure containing the channel, the bionic structure Store at low temperature, or continue in vitro culture or directly for organ transplantation.
在上述电场、磁场和电磁复合场训练仿生结构的方法的基础上，本发明还将脉动培养系统同时应用其中的训练方法，其步骤包括上述步骤，此外还包括脉动培养系统的引入和使用：启动脉动培养系统701，所述脉动培养系统701包括脉动系统电机702、脉动系统导轨-滑块机构703、培养液供给注射器704、单向阀705、导流管706和培养液瓶708；所述脉动系统电机702通过曲柄与脉动系统导轨-滑块机构703连接；所述导轨-滑块机构703与培养液供给注射器704连接；通过导流管706将所述仿生结构与脉动培养系统连接；使培养液在导流管706和含通道的仿生结构之间进行单向流动；液体流速为大于0，小于30cm/s，进行脉动-电场、脉动-磁场或脉动-电磁复合场的作用；完成对所述仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。Based on the above methods of training the bionic structure of the electric field, the magnetic field and the electromagnetic composite field, the present invention also applies the training method to the pulsation culture system, the steps including the above steps, and the introduction and use of the pulsation culture system: starting a pulsation culture system 701, the pulsation culture system 701 includes a pulsation system motor 702, a pulsation system guide-slider mechanism 703, a culture fluid supply syringe 704, a check valve 705, a draft tube 706, and a culture fluid bottle 708; The system motor 702 is coupled to the pulsation system rail-slider mechanism 703 by a crank; the rail-slider mechanism 703 is coupled to the culture fluid supply injector 704; the biomimetic structure is coupled to the pulsation culture system via a flow conduit 706; The liquid flows in a one-way flow between the draft tube 706 and the bionic structure containing the channel; the liquid flow rate is greater than 0, less than 30 cm/s, and the action of the pulsation-electric field, the pulsation-magnetic field or the pulsation-electromagnetic composite field is performed; After the training of the biomimetic structure, the biomimetic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
下面举出几个具体的实施例，以进一步理解本发明。Several specific embodiments are set forth below to further understand the present invention.
实施例1：利用多喷头3D打印法制备一种含血管和神经系统双通道的仿心脏结构，并将该仿心脏结构在电场中进行训练。Example 1: A pseudo-heart structure containing two channels of blood vessels and nervous systems was prepared by multi-nozzle 3D printing, and the pseudo-heart structure was trained in an electric field.
a)利用计算机设计所述含多通道的仿生心脏结构的三维模型；a) using a computer to design a three-dimensional model of the multi-channel bionic heart structure;
b)将心肌细胞作为主体细胞，与配置好的海藻酸钠溶液混合，海藻酸钠水凝胶溶液质量体积浓度为0.5％；之后将该混合物装载到多喷头3D打印设备的一个喷头组件中；b) the cardiomyocytes are used as host cells, mixed with the configured sodium alginate solution, and the sodium alginate hydrogel solution has a mass volume concentration of 0.5%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
c)将血管内皮细胞和神经元作为通道细胞；将血管内皮细胞与海藻酸钠溶液混合，装入3D打印设备的第二个喷头组件中；将神经元与细胞培养液(DMEM)混合，装入3D打印设备的第三个喷头组件中； c) using vascular endothelial cells and neurons as channel cells; mixing vascular endothelial cells with sodium alginate solution into a second nozzle assembly of a 3D printing device; mixing neurons with cell culture fluid (DMEM), loading Into the third nozzle assembly of the 3D printing device;
d)由计算机控制多喷头3D打印设备的不同喷头组件，控制主体结构的心肌细胞/海藻酸钠的成形；同步或异步控制血管内皮细胞/海藻酸钠的通道成形，得到血管系统通道；同步或异步控制神经元/DMEM，将其定位与通道孔内，得到神经系统通道；并采用CaCl2使海藻酸钠溶液交联得到海藻酸钠水凝胶，逐层堆积，得到所述含多通道的双通道的仿心脏结构；d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of myocardial cells / sodium alginate in the main structure; synchronous or asynchronous control of vascular endothelial cells / sodium alginate channel formation, get vascular system channels; synchronization or Asynchronously control the neurons/DMEM, locate them in the channel pores, and obtain the nervous system channels; and cross-link the sodium alginate solution with CaCl2 to obtain a sodium alginate hydrogel, which is stacked layer by layer to obtain the multi-channel double The simulated heart structure of the channel;
e)将制备好的仿心脏结构至于电磁训练装置的样品台上；e) placing the prepared simulated heart structure on the sample stage of the electromagnetic training device;
f)将电磁训练装置的正极和负极的金属平板仿心脏结构的两侧但不接触；f) the metal plates of the positive and negative electrodes of the electromagnetic training device are imitated on both sides of the heart structure but are not in contact;
g)将将电磁训练装置的电场发生装置通电，并由控制电压大小为直流50V；g) The electric field generating device of the electromagnetic training device will be energized, and the control voltage is DC 50V;
h)完成电场对仿心脏结构的训练后，将其继续进行体外培养。h) After completing the training of the electric field on the simulated heart structure, it is continued to be cultured in vitro.
实施例2：利用多喷头3D打印法制备一种含血管、神经和免疫系统三通道的仿肾脏结构，并将该仿肾脏结构在电场中进行训练。Example 2: A multi-nozzle 3D printing method was used to prepare a pseudo-renal structure containing three channels of blood vessels, nerves and immune system, and the simulated kidney structure was trained in an electric field.
a)利用计算机设计所述含多通道的仿生心脏结构的三维模型；a) using a computer to design a three-dimensional model of the multi-channel bionic heart structure;
b)将胚肾细胞作为主体细胞，与配置好的纤维蛋白原溶液混合，纤维蛋白原溶液质量体积浓度为3％；之后将该混合物装载到多喷头3D打印设备的一个喷头组件中；b) the embryonic kidney cells are used as host cells, mixed with the configured fibrinogen solution, and the fibrinogen solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
c)将血管内皮细胞、平滑肌细胞、神经元和T淋巴细胞作为通道细胞；将血管内皮细胞和平滑肌细胞与海藻酸钠溶液混合，装入3D打印设备的第二个喷头组件中；将神经元与海藻酸钠溶液混合，装入3D打印设备的第三个喷头组件中；将T淋巴细胞与海藻酸钠溶液混合，装入3D打印设备的第四个喷头组件中；c) using vascular endothelial cells, smooth muscle cells, neurons, and T lymphocytes as channel cells; mixing vascular endothelial cells and smooth muscle cells with sodium alginate solution, and loading them into a second nozzle assembly of the 3D printing device; Mixing with the sodium alginate solution, loading into the third nozzle assembly of the 3D printing device; mixing the T lymphocytes with the sodium alginate solution into the fourth nozzle assembly of the 3D printing device;
d)由计算机控制多喷头3D打印设备的不同喷头组件，控制主体结构的胚肾细胞/纤维蛋白原的成形，并采用凝血酶使纤维蛋白原凝胶化；同步或异步控制毛细血管内皮细胞和平滑肌细胞/海藻酸钠的通道成形，得到血管系统通道；同步或异步控制神经元/海藻酸钠的通道成形，得到神经系统通道；同步或异步控制T淋巴细胞/海藻酸钠的通道成形，得到免疫系统通道；采用CaCl2使海藻酸钠溶液凝胶化得到海藻酸钠水凝胶，逐层堆积，得到所述含多通道的三通道的仿肾脏结构。d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of embryonic kidney cells / fibrinogen of the main structure, and gelatinization of fibrinogen by thrombin; synchronous or asynchronous control of capillary endothelial cells and Channel formation of smooth muscle cells/sodium alginate to obtain vascular system channels; synchronous or asynchronous control of neuronal/sodium alginate channel formation to obtain neural system channels; simultaneous or asynchronous control of T lymphocyte/sodium alginate channel formation, Immune system channel; sodium alginate hydrogel was gelated by CaCl 2 to obtain sodium alginate hydrogel, and stacked layer by layer to obtain the multi-channel three-channel imitation kidney structure.
e)将制备好的仿肾脏结构至于电磁训练装置的样品台上；e) preparing the prepared kidney structure onto the sample stage of the electromagnetic training device;
f)将电磁训练装置的正极和负极的金属探针与仿肾脏结构连接，使仿肾脏结构成为电路一部分；f) connecting the metal probes of the positive and negative electrodes of the electromagnetic training device with the imitation kidney structure, so that the imitation kidney structure becomes a part of the circuit;
g)将将电磁训练装置的电场发生装置通电，并由控制电流大小为直流50mA；g) The electric field generating device of the electromagnetic training device will be energized, and the control current is DC 50 mA;
h)完成电场对仿肾脏结构的训练后，将其继续进行体外培养。h) After completing the training of the imitation kidney structure by the electric field, continue to culture in vitro.
实施例3：利用多喷头3D打印法制备一种含一进一出血管、神经、胆管和免疫系统五通道的仿肝脏结构，并将该仿肝脏结构在磁场中进行训练。Example 3: A multi-nozzle 3D printing method was used to prepare a liver-like structure containing five channels of blood vessels, nerves, bile ducts, and immune system, and the liver-like structure was trained in a magnetic field.
a)利用计算机设计所述含多通道的仿生心脏结构的三维模型；a) using a computer to design a three-dimensional model of the multi-channel bionic heart structure;
b)将脂肪干细胞和肝细胞作为主体细胞，与配置好的明胶、明胶/纤维蛋白原溶液混合，明胶、明胶/纤维蛋白原溶液质量体积浓度为分别为20％、10％；之后将该混合物装载到多喷头3D打印设备的一个喷头组件中； b) using adipose stem cells and hepatocytes as host cells, mixed with the gelatin and gelatin/fibrinogen solution, and the mass and volume concentrations of the gelatin and gelatin/fibrinogen solutions are 20% and 10%, respectively; Loading into a showerhead assembly of a multi-nozzle 3D printing device;
c)将血管内皮细胞和B淋巴细胞作为通道细胞(这两种细胞已与Fe3O4磁性纳米颗粒混合)；将血管内皮细胞与胶原溶液混合，装入3D打印设备的第二个喷头组件中；将B淋巴细胞与胶原溶液混合，装入3D打印设备的第三个喷头组件中；c) using vascular endothelial cells and B lymphocytes as channel cells (both of which have been mixed with Fe 3 O 4 magnetic nanoparticles); mixing vascular endothelial cells with collagen solution and loading the second nozzle assembly of the 3D printing device Mixing B lymphocytes with a collagen solution and loading it into a third nozzle assembly of the 3D printing device;
d)由计算机控制多喷头3D打印设备的不同喷头组件，控制主体结构脂肪干细胞/明胶和肝细胞/明胶/纤维蛋白原的成形，并采用凝血酶使纤维蛋白原聚合；同步或异步控制血管内皮细胞/胶原的通道成形，得到两个血管通道；同步或异步控制B淋巴细胞/胶原的通道成形，得到免疫系统通道；采用戊二醛溶液使胶原交联为水凝胶，逐层堆积，得到所述含多通道的三通道的仿肝脏结构。d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of the main structure of adipose stem cells / gelatin and hepatocytes / gelatin / fibrinogen, and use thrombin to polymerize fibrinogen; synchronous or asynchronous control of vascular endothelium Cell/collagen channel formation, obtaining two vascular channels; synchronous or asynchronous control of B lymphocyte/collagen channel formation, obtaining immune system channels; using glutaraldehyde solution to crosslink collagen into hydrogel, layer by layer, The multi-channel, three-channel, liver-like structure.
e)将制备好的带磁性的仿肝脏结构至于电磁训练装置的样品台上；e) preparing the magnetically imitation liver structure onto the sample stage of the electromagnetic training device;
f)由电机驱动控制磁场发生系统的转动、磁场大小和作用时间，使得磁感线穿过带磁性的仿肝脏结构；f) controlling the rotation of the magnetic field generating system, the magnitude of the magnetic field and the action time by the motor drive, so that the magnetic induction line passes through the magnetic liver-like structure;
g)完成磁场对仿肝脏生结构的训练后，将其继续进行体外培养。g) After completing the training of the magnetic field on the liver-like structure, continue to culture in vitro.
实施例4：利用多内芯旋转组合模具法制备一种含一进一出双血管和神经系统三通道的仿胰腺结构，并将该仿生胰腺组织在复合电磁场中进行训练。Example 4: A pancreatic structure containing one-in-one-out dual-vessel and three-channel nervous system was prepared by a multi-core rotation combined mold method, and the bionic pancreatic tissue was trained in a composite electromagnetic field.
a)将胰岛β细胞作为主体细胞，与配置好的纤维蛋白原溶液混合，该混合物中纤维蛋白原溶液质量体积浓度为5％；a) the islet β cells as a host cell, mixed with the configured fibrinogen solution, the mass concentration of the fibrinogen solution in the mixture is 5%;
b)将脂肪干细胞和雪旺氏细胞作为通道细胞，与细胞培养基(DMEM)混合；b) using adipose stem cells and Schwann cells as channel cells, mixed with cell culture medium (DMEM);
c)将三个模具分支内芯插入模具底座的阵列孔洞中，将顶模具套在模具底座上，并使模具分支内芯穿过顶模具的上部中央的大孔；c) inserting the inner cores of the three mold branches into the array holes of the mold base, placing the top mold on the mold base, and passing the core of the mold branch through the large hole in the upper center of the top mold;
d)将步骤胰岛β细胞/纤维蛋白原混合物通过大孔灌注到顶模具的内腔中，同时使模具底座与顶模具产生相对旋转，旋转速度为30r/min，模具底座与模具分支内芯相对静止，使灌注的混合物形成半纺锤外形，旋转过程中采用凝血酶使纤维蛋白原转变为水凝胶；d) injecting the step islet β cell/fibrinogen mixture into the inner cavity of the top mold through the macropores, and simultaneously rotating the mold base and the top mold at a rotation speed of 30 r/min, and the mold base and the mold branch core are relatively stationary. , the perfused mixture forms a semi-spindle shape, and thrombin is used to convert fibrinogen into a hydrogel during rotation;
e)依次去除顶模具和模具分支内芯，形成含三通道仿生结构前体；e) sequentially removing the top mold and the mold branch inner core to form a three-channel biomimetic structure precursor;
f)将脂肪干细胞/DMEM灌注到三通道仿生结构前体的一进一出双管道中，形成血管系统通道；将雪旺氏细胞/DMEM灌注到通道仿生结构前体的另一个通道中，形成神经系统通道；最终，得到三通道的仿胰腺结构。f) infusing adipose stem cells/DMEM into one-in-one double-tube of a three-channel biomimetic structure precursor to form a vascular system channel; injecting Schwann cells/DMEM into another channel of the channel biomimetic structure precursor to form The nervous system channel; eventually, a three-channel pancreatic structure is obtained.
g)将制备好的仿胰腺结构至于电磁训练装置的样品台上；g) preparing the prepared pancreatic structure onto the sample stage of the electromagnetic training device;
h)将电磁训练装置的正极和负极的金属探针与仿胰腺结构连接，使仿胰腺结构成为电路一部分；h) connecting the metal probes of the positive and negative electrodes of the electromagnetic training device with the pancreatic structure, so that the pancreatic structure becomes part of the circuit;
i)将将电磁训练装置的电场发生装置通电，并由控制电流大小、方向和作用时间；电场线此时穿过仿胰腺结构；i) energizing the electric field generating device of the electromagnetic training device, and controlling the magnitude, direction and duration of the current; the electric field line passes through the pancreatic structure at this time;
j)由电机驱动控制磁场发生系统的转动、磁场大小和作用时间，使得磁感线也穿过的仿胰腺结构；j) by the motor drive to control the rotation of the magnetic field generating system, the size of the magnetic field and the action time, so that the magnetic induction line also passes through the pancreatic structure;
k)完成复合电场磁场对仿胰腺结构的训练后，将其继续进行体外培养。 k) After completing the training of the pseudo-pancreatic structure by the composite electric field magnetic field, it is further cultured in vitro.
实施例5：利用多内芯旋转组合模具法制备一种含血管、胆管、神经和免疫系统四通道的肝脏结构，并将该肝脏结构在复合电磁场中进行训练。Example 5: A liver structure containing four channels of blood vessels, bile ducts, nerves and immune system was prepared by a multi-core rotating combination mold method, and the liver structure was trained in a composite electromagnetic field.
a)将肝细胞和脂肪干作为主体细胞，与配置好的海藻酸钠/明胶溶液、纤维蛋白原溶液混合，该混合物中海藻酸钠、纤维蛋白原质量体积浓度为5％；在该混合物中加入质量体积浓度3％的DMSO；a) using hepatocytes and adipose stem as host cells, mixed with a configured sodium alginate/gelatin solution, fibrinogen solution, wherein the mixture has a volume concentration of sodium alginate and fibrinogen of 5%; in the mixture Adding 3% by volume of DMSO;
b)将内皮细胞、雪旺氏细胞和中性粒细胞作为通道细胞；将内皮细胞和雪旺氏细胞与细胞培养基(DMEM)混合；将中性粒细胞与海藻酸钠溶液混合；b) using endothelial cells, Schwann cells, and neutrophils as channel cells; mixing endothelial cells and Schwann cells with cell culture medium (DMEM); mixing neutrophils with sodium alginate solution;
c)将四个模具分支内芯插入模具底座的阵列孔洞中，将顶模具套在模具底座上，并使模具分支内芯穿过顶模具的上部中央的大孔；c) inserting four mold branch cores into the array holes of the mold base, sleeve the top mold on the mold base, and passing the mold branch inner core through the large hole in the upper center of the top mold;
d)将肝细胞与脂肪干细胞的纤维蛋白原/DMSO溶液、海藻酸钠/明胶/DMSO溶液通过大孔灌注到顶模具的内腔中，同时使模具底座与顶模具产生相对旋转，旋转速度为30r/min，模具底座与模具分支内芯相对静止，使灌注的混合物形成半纺锤外形，旋转过程中采用CaCl2使海藻酸钠转变为水凝胶；d) injecting fibrinogen/DMSO solution of adipose stem cells and adipose stem cells, sodium alginate/gelatin/DMSO solution into the inner cavity of the top mold through a large hole, and simultaneously rotating the mold base and the top mold at a rotation speed of 30 r /min, the mold base and the core of the mold branch are relatively stationary, so that the poured mixture forms a semi-spindle shape, and the sodium alginate is converted into a hydrogel by using CaCl 2 during the rotation;
e)依次去除顶模具和模具分支内芯，形成含四通道仿生结构前体；e) sequentially removing the top mold and the mold branch inner core to form a four-channel biomimetic structure precursor;
f)将脂肪干细胞/肝素/EGF/PBS悬浮液灌注到四通道仿生结构前体的第一个通道中，形成血管系统通道；将雪旺氏细胞PBS悬浮液灌注到四通道仿生结构前体的第二个通道中，形成神经系统通道；将中性粒细胞/PBS悬浮液灌注到四通道仿生结构前体的第三个通道中，并采用CaCl2交联，形成免疫系统通道；最终，得到四通道的半纺锤形肝脏结构。将两个半纺锤形肝脏前体用海藻酸钠/明胶/DMSO溶液粘附在一起，外喷一层合成高分子聚氨酯溶液，形成一个纺锤形肝脏前体；f) perfusion of adipose stem cell/heparin/EGF/PBS suspension into the first channel of a four-channel biomimetic structure precursor to form a vascular system channel; perfusion of Schwann cell PBS suspension into a four-channel biomimetic structure precursor In the second channel, the nervous system channel is formed; the neutrophil/PBS suspension is perfused into the third channel of the four-channel biomimetic structure precursor, and CaCl 2 is cross-linked to form the immune system channel; finally, Four-channel semi-spindle liver structure. The two semi-spindle liver precursors are adhered together with a sodium alginate/gelatin/DMSO solution, and a layer of synthetic high molecular polyurethane solution is sprayed to form a spindle-shaped liver precursor;
g)将制备好的纺锤形肝脏前体至于电磁训练装置的样品台上；g) preparing the prepared spindle-shaped liver precursor onto the sample stage of the electromagnetic training device;
h)将电磁训练装置的正极和负极的金属平板置于纺锤形肝脏前体的两侧但不接触；h) placing the metal plates of the positive and negative electrodes of the electromagnetic training device on both sides of the spindle-shaped liver precursor without contact;
i)将将电磁训练装置的电场发生装置通电，并由控制电压为直流20V，电场线此时穿过纺锤形肝脏前体结构；i) energizing the electric field generating device of the electromagnetic training device, and the control voltage is 20 V DC, and the electric field line passes through the spindle-shaped liver precursor structure at this time;
j)由电机驱动控制磁场发生系统的转动、磁场大小和作用时间，使得磁感线也穿过的纺锤形肝脏前体结构；j) a spindle-shaped liver precursor structure that is driven by a motor to control the rotation of the magnetic field generating system, the magnitude of the magnetic field, and the action time so that the magnetic induction line also passes;
k)完成复合电场磁场对纺锤形肝脏前体的训练后，将其放入液氮中保存，临床需要时复苏移植。k) After completing the training of the composite electric field magnetic field on the spindle-shaped liver precursor, it is stored in liquid nitrogen, and the transplant is resuscitated when clinically needed.
实施例6：利用多内芯旋转组合模具法制备一种含血管、神经和免疫系统四通道的心脏结构，并将该心脏结构在电场中进行训练。Example 6: A four-channel heart structure containing blood vessels, nerves, and immune system was prepared using a multi-core rotating combination mold method, and the heart structure was trained in an electric field.
a)将心肌细胞作为主体细胞，与配置好的纤维蛋白原溶液混合，该混合物中纤维蛋白原溶液质量体积浓度为5％；在该混合物中加入质量体积浓度3％的右旋糖；a) the cardiomyocytes as a host cell, mixed with the configured fibrinogen solution, the mass concentration of the fibrinogen solution in the mixture is 5%; adding a mass volume concentration of 3% dextrose to the mixture;
b)将脂肪干细胞、平滑肌细胞、神经元和和淋巴细胞作为通道细胞，分别与细胞培养基(DMEM)混合； b) using adipose stem cells, smooth muscle cells, neurons, and lymphocytes as channel cells, respectively, mixed with cell culture medium (DMEM);
c)将四个模具分支内芯插入模具底座的阵列孔洞中，将顶模具套在模具底座上，并使模具分支内芯穿过顶模具的上部中央的大孔；c) inserting four mold branch cores into the array holes of the mold base, sleeve the top mold on the mold base, and passing the mold branch inner core through the large hole in the upper center of the top mold;
d)将心肌/纤维蛋白原/右旋糖溶液混合物通过大孔灌注到顶模具的内腔中，同时使模具底座与顶模具产生相对旋转，旋转速度为30r/min，模具底座与模具分支内芯相对静止，使灌注的混合物形成半纺锤外形，旋转过程中采用凝血酶使纤维蛋白原转变为水凝胶；d) The myocardial/fibrinogen/dextrose solution mixture is perfused through the macropores into the inner cavity of the top mold, while the mold base and the top mold are relatively rotated, the rotation speed is 30r/min, and the mold base and the mold branch core are Relatively static, the perfused mixture forms a semi-spindle shape, and thrombin is used to convert fibrinogen to hydrogel during rotation;
e)依次去除顶模具和模具分支内芯，形成含四通道仿生结构前体；e) sequentially removing the top mold and the mold branch inner core to form a four-channel biomimetic structure precursor;
f)将脂肪干细胞/EGF/b-FGF/紫杉醇/DMEM灌注到四通道仿生结构前体的第一个通道中，形成血管系统通道；将脂肪干细胞和平滑肌细胞/DMEM灌注到四通道仿生结构前体的第二个通道中，形成血管系统通道；将神经元/DMEM灌注到四通道仿生结构前体的第三个通道中，形成神经系统通道；将淋巴细胞/DMEM灌注到四通道仿生结构前体的第四个通道中，形成免疫系统淋巴通道；最终，得到四通道的仿心脏结构。f) perfusion of adipose stem cells/EGF/b-FGF/paclitaxel/DMEM into the first channel of a four-channel biomimetic structure precursor to form a vascular system channel; perfusion of adipose stem cells and smooth muscle cells/DMEM into a four-channel biomimetic structure In the second channel of the body, a vascular system channel is formed; the neuron/DMEM is perfused into the third channel of the four-channel biomimetic structure precursor to form a nervous system channel; before the lymphocyte/DMEM is perfused into the four-channel biomimetic structure In the fourth channel of the body, the lymphatic passage of the immune system is formed; finally, a four-channel pseudo-cardiac structure is obtained.
g)将制备好的仿心脏结构至于电磁训练装置的样品台上；g) preparing the prepared simulated cardiac structure on the sample stage of the electromagnetic training device;
h)将电磁训练装置的正极和负极的金属导线与仿心脏结构连接，使心脏结构成为电路一部分；h) connecting the metal wires of the positive and negative electrodes of the electromagnetic training device with the pseudo-cardiac structure, so that the cardiac structure becomes part of the circuit;
i)将将电磁训练装置的电场发生装置通电，并由控制电流大小直流交流交替30mA；i) the electric field generating device of the electromagnetic training device will be energized, and the current is controlled by a direct current alternating current of 30 mA;
j)完成电场对心脏结构的训练后，将其继续进行体外培养。j) After completing the training of the electric field on the cardiac structure, it is continued to be cultured in vitro.
实施例7：利用多通道可拆分组合模具法制备一种含血管、神经和免疫系统多通道的人工皮肤结构，并将该皮肤结构在电磁场中进行训练。Example 7: An artificial skin structure containing multiple channels of blood vessels, nerves, and immune system was prepared using a multi-channel detachable combination mold method, and the skin structure was trained in an electromagnetic field.
a)将成纤维细胞作为主体细胞，与配置好的海藻酸钠溶液混合，该混合物中海藻酸钠溶液质量体积浓度为5％；a) the fibroblasts as a host cell, mixed with the configured sodium alginate solution, the mass concentration of the sodium alginate solution in the mixture is 5%;
b)将成纤维细胞、脂肪干细胞作为主体细胞，神经元和和淋巴细胞作为通道细胞，角质细胞、上皮细胞作为复合细胞，分别于细胞培养基(DMEM)混合；b) fibroblasts, adipose stem cells as host cells, neurons and lymphocytes as channel cells, keratinocytes, epithelial cells as composite cells, respectively mixed in cell culture medium (DMEM);
c)将多通道可拆分模具插入模具底座的阵列孔洞中，将外模具套在模具底座上；c) inserting a multi-channel detachable mold into the array hole of the mold base, and placing the outer mold on the mold base;
d)将成纤维细胞/海藻酸钠溶液混合物灌注到顶模具的内腔中，使灌注的混合物形成正方形，采用CaCl2使海藻酸钠转变为水凝胶；d) injecting a fibroblast/sodium alginate solution mixture into the lumen of the top mold, forming a square of the perfused mixture, and converting the sodium alginate to a hydrogel using CaCl 2 ;
e)依次去除外模具和模具分支内芯，形成含多通道人工皮肤仿生结构前体；e) sequentially removing the outer mold and the inner core of the mold branch to form a precursor comprising a multi-channel artificial skin bionic structure;
f)将脂肪干细胞/DMEM灌注到四通道仿生结构前体的第一个通道中，形成血管系统通道；将脂肪干细胞和平滑肌细胞/DMEM灌注到四通道仿生结构前体的第二个通道中，形成血管系统通道；将神经元/DMEM灌注到四通道仿生结构前体的第三个通道中，形成神经系统通道；将淋巴细胞/DMEM灌注到四通道仿生结构前体的第四个通道中，形成免疫系统淋巴通道；最终，得到四通道的人工皮肤结构。f) infusing adipose stem cells/DMEM into the first channel of a four-channel biomimetic structure precursor to form a vascular system channel; infusing adipose stem cells and smooth muscle cells/DMEM into a second channel of a four-channel biomimetic structure precursor, Forming a vascular system channel; perfusing the neuron/DMEM into the third channel of the four-channel biomimetic structure precursor to form a nervous system channel; perfusing the lymphocyte/DMEM into the fourth channel of the four-channel biomimetic structure precursor, The lymphatic passage of the immune system is formed; eventually, a four-channel artificial skin structure is obtained.
g)将制备好的人工皮肤结构至于电磁训练装置的样品台上；g) preparing the artificial skin structure onto the sample stage of the electromagnetic training device;
h)将电磁训练装置的正极和负极的金属导线与人工皮肤结构连接，使人工皮肤结构成为电路一部分； h) connecting the metal wires of the positive and negative electrodes of the electromagnetic training device to the artificial skin structure, so that the artificial skin structure becomes part of the circuit;
i)将将电磁训练装置的电场发生装置通电，并由控制电流大小为交流20mA；i) the electric field generating device of the electromagnetic training device will be energized, and the control current is AC 20 mA;
j)完成电场对人工皮肤结构的训练后，将其继续进行体外培养。j) After completing the training of the electric field on the artificial skin structure, it is continued to be cultured in vitro.
实施例8：利用多内芯旋转组合模具法制备一种含血管、神经和免疫系统三通道的乳房结构，并将该乳房结构在复合电磁场中进行训练。Example 8: A three-channel breast structure containing blood vessels, nerves and immune system was prepared by a multi-core rotation combined mold method, and the breast structure was trained in a composite electromagnetic field.
a)将脂肪干细胞作为主体细胞，与配置好的海藻酸钠溶液混合，该混合物中海藻酸钠溶液质量体积浓度为1％；a) the adipose stem cells as a host cell, mixed with the configured sodium alginate solution, the mass concentration of the sodium alginate solution in the mixture is 1%;
b)将内皮细胞、雪旺氏细胞和中性粒细胞作为通道细胞；将内皮细胞和雪旺氏细胞与细胞培养基(DMEM)混合；将中性粒细胞与海藻酸钠溶液混合；b) using endothelial cells, Schwann cells, and neutrophils as channel cells; mixing endothelial cells and Schwann cells with cell culture medium (DMEM); mixing neutrophils with sodium alginate solution;
c)将三个模具分支内芯插入模具底座的阵列孔洞中，将顶模具套在模具底座上，并使模具分支内芯穿过顶模具的上部中央的大孔；c) inserting the inner cores of the three mold branches into the array holes of the mold base, placing the top mold on the mold base, and passing the core of the mold branch through the large hole in the upper center of the top mold;
d)将脂肪干细胞/海藻酸钠/DMSO溶液混合物通过大孔灌注到顶模具的内腔中，同时使模具底座与顶模具产生相对旋转，旋转速度为30r/min，模具底座与模具分支内芯相对静止，使灌注的混合物形成半纺锤外形，旋转过程中采用CaCl2使海藻酸钠转变为水凝胶；d) The adipose stem cell/sodium alginate/DMSO solution mixture is poured into the inner cavity of the top mold through the macropores, and the mold base and the top mold are relatively rotated at a rotation speed of 30 r/min, and the mold base is opposite to the mold core. Static, so that the perfused mixture forms a semi-spindle shape, and the sodium alginate is converted into a hydrogel by using CaCl 2 during the rotation;
e)依次去除顶模具和模具分支内芯，形成含三通道仿生结构前体；e) sequentially removing the top mold and the mold branch inner core to form a three-channel biomimetic structure precursor;
f)将内皮细胞/DMEM灌注到三通道仿生结构前体的第一个通道中，形成血管系统通道；将雪旺氏细胞/DMEM灌注到三通道仿生结构前体的第二个通道中，形成神经系统通道；将中性粒细胞/海藻酸钠溶液灌注到三通道仿生结构前体的第三个通道中，并采用CaCl2交联，形成免疫系统通道；最终，得到三通道的半纺锤形乳房结构。将两个半纺锤形乳房前体用海纤维蛋白原/脂肪干细胞溶液粘附在一起，外喷一层合成高分子聚乳酸和聚羟基乙酸共聚物(PLGA)溶液，形成一个纺锤形乳房前体；f) perfusion of endothelial cells/DMEM into the first channel of the three-channel biomimetic structure precursor to form a vascular system channel; injecting Schwann cells/DMEM into the second channel of the three-channel biomimetic structure precursor to form The nervous system channel; the neutrophil/sodium alginate solution is infused into the third channel of the three-channel biomimetic structure precursor, and cross-linked with CaCl 2 to form the immune system channel; finally, a three-channel semi-spindle shape is obtained. Breast structure. The two semi-spindle breast precursors are adhered together with a fibrinogen/adipose stem cell solution, and a layer of synthetic high molecular polylactic acid and polyglycolic acid copolymer (PLGA) solution is sprayed to form a spindle-shaped breast precursor. ;
g)将制备好的纺锤形乳房前体至于电磁训练装置的样品台上；g) preparing the prepared spindle-shaped breast precursor onto the sample stage of the electromagnetic training device;
h)将电磁训练装置的正极和负极的金属平板置于纺锤形乳房前体的两侧但不接触；h) placing the metal plates of the positive and negative electrodes of the electromagnetic training device on both sides of the spindle-shaped breast precursor without contact;
i)将将电磁训练装置的电场发生装置通电，并由控制电流大小、方向和作用时间；电场线此时穿过纺锤形乳房前体结构；i) energizing the electric field generating device of the electromagnetic training device, and controlling the magnitude, direction and duration of the current; the electric field line passes through the spindle-shaped breast precursor structure at this time;
j)由电机驱动控制磁场发生系统的转动、磁场大小和作用时间，使得磁感线也穿过的纺锤形乳房前体结构；j) a spindle-shaped breast precursor structure that controls the rotation of the magnetic field generating system, the magnitude of the magnetic field, and the action time by the motor so that the magnetic induction line also passes;
k)完成复合电场磁场对纺锤形乳房前体的训练后，将其放入液氮中保存，临床需要时复苏移植。k) After completing the training of the composite electric field magnetic field on the spindle-shaped breast precursor, it is stored in liquid nitrogen, and the transplant is resuscitated when clinically needed.
实施例9：利用组合模具法制备一种含两进两出血管、神经和免疫系统四通道的肺叶结构，并将该肺叶结构在磁场中进行训练。Example 9: A lung lobe structure containing two channels of blood vessels, nerves, and immune system was prepared by a combined mold method, and the lung lobe structure was trained in a magnetic field.
a)利用计算机设计所述含四通道的仿生肺叶结构的三维模型与组合模具；a) using a computer to design a three-dimensional model and a combined mold of the four-channel bionic lung leaf structure;
b)将脂肪干细胞和肺上皮细胞作为主体细胞，与配置好的明胶溶液混合，明胶溶液质量体积浓度为分别为10％，其中加入质量分数为3％的细胞冻存剂甘油，之后将该混合物注射 到组合模具的相应位置，然后将1％胶原溶液注射或灌注在相应的细胞层上，37℃下静止30分钟使细胞明胶和胶原层固化，或采用戊二醛溶液使胶原转变为凝胶；b) Adipose stem cells and lung epithelial cells are used as host cells, and mixed with the prepared gelatin solution, the mass concentration of the gelatin solution is 10%, respectively, and the cell cryopreservative glycerin is added in a mass fraction of 3%, and then the mixture is added. Injection To the corresponding position of the combined mold, then inject or infuse the 1% collagen solution onto the corresponding cell layer, freeze the cell gelatin and collagen layer at 37 ° C for 30 minutes, or convert the collagen into a gel using a glutaraldehyde solution;
c)将血管内皮细胞和B淋巴细胞作为通道细胞(这两种细胞已与Fe3O4磁性纳米颗粒混合)；将血管内皮细胞与纤维蛋白原溶液混合，注射到两进两出的血管系统中；将B淋巴细胞与纤维蛋白原溶液混合，注射到一个分支管道系统中；将雪旺细胞与纤维蛋白原溶液混合注射到另一个分支神经系统中；c) using vascular endothelial cells and B lymphocytes as channel cells (both of which have been mixed with Fe 3 O 4 magnetic nanoparticles); mixing vascular endothelial cells with fibrinogen solution and injecting into the two-in and two-out vascular system Mixing B lymphocytes with fibrinogen solution and injecting into a branching system; mixing Schwann cells with fibrinogen solution into another branching nervous system;
d)采用0.1％凝血酶溶液使纤维蛋白原为水凝胶，得到四通道的肺叶结构。d) Fibrinogen was hydrogeled with 0.1% thrombin solution to obtain a four-channel lobes structure.
e)将制备好的含细胞冻存剂的多通道样品置于样品台上，将多通道样品的血管系统与脉动生物反应器相连；e) placing a prepared multi-channel sample containing cell cryopreservation on the sample stage, and connecting the vascular system of the multi-channel sample to the pulsating bioreactor;
f)控制脉动生物反应器中培养液的流量为30cm/s，作用时间3h，使其中细胞形成组织结构；f) controlling the flow rate of the culture solution in the pulsating bioreactor to be 30 cm/s, and the action time is 3 hours, so that the cells form a tissue structure;
g)完成脉动生物反应器对肺叶结构的力学训练后，将其放在培养液中继续培养，或放在低温(如液氮中)长期保存，或直接用于器官移植。g) After completing the mechanical training of the pulsating bioreactor on the structure of the lung lobe, it is placed in the culture medium to continue the culture, or placed in a low temperature (such as liquid nitrogen) for long-term storage, or directly used for organ transplantation.
实施例10：利用多喷头3D打印法制备一种含血管和神经系统双通道的仿肝肿瘤结构，并将该仿肝肿瘤结构在电场中进行脉动培养训练。Example 10: A liver-like tumor structure containing a vascular and a nervous system dual channel was prepared by a multi-nozzle 3D printing method, and the liver-like tumor structure was subjected to pulsation culture training in an electric field.
a)利用计算机设计所述含多通道的仿生肝肿瘤结构的三维模型；a) using a computer to design a three-dimensional model of the multi-channel biomimetic liver tumor structure;
b)将肝癌细胞作为主体细胞，与配置好的海藻酸钠溶液混合，海藻酸钠水凝胶溶液质量体积浓度为3％；之后将该混合物装载到多喷头3D打印设备的一个喷头组件中；b) the liver cancer cells are used as host cells, and mixed with the configured sodium alginate solution, the sodium alginate hydrogel solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
c)将血管内皮细胞和神经元作为通道细胞；将血管内皮细胞与海藻酸钠溶液混合，装入3D打印设备的第二个喷头组件中；将神经元与细胞培养液(DMEM)混合，装入3D打印设备的第三个喷头组件中；c) using vascular endothelial cells and neurons as channel cells; mixing vascular endothelial cells with sodium alginate solution into a second nozzle assembly of a 3D printing device; mixing neurons with cell culture fluid (DMEM), loading Into the third nozzle assembly of the 3D printing device;
d)由计算机控制多喷头3D打印设备的不同喷头组件，控制主体结构的肝癌细胞/海藻酸钠的成形；同步或异步控制血管内皮细胞/海藻酸钠的通道成形，得到血管系统通道；同步或异步控制神经元/DMEM，将其定位与通道孔内，得到神经系统通道；并采用CaCl2使海藻酸钠溶液交联得到海藻酸钠水凝胶，逐层堆积，得到所述含多通道的双通道的仿肝肿瘤结构；d) computer controlled multi-nozzle 3D printing device different nozzle components, control of the formation of liver cancer cells / sodium alginate; synchronous or asynchronous control of vascular endothelial cells / sodium alginate channel formation, get vascular system channels; synchronization or Asynchronously control the neurons/DMEM, locate them in the channel pores, and obtain the nervous system channels; and cross-link the sodium alginate solution with CaCl 2 to obtain a sodium alginate hydrogel, which is stacked layer by layer to obtain the multi-channel-containing Dual-channel liver-like tumor structure;
e)将制备好的仿肝肿瘤结构至于电磁训练装置的样品台上；e) placing the prepared liver-like tumor structure on the sample stage of the electromagnetic training device;
f)将电磁训练装置的正极和负极的金属平板仿心脏结构的两侧但不接触；f) the metal plates of the positive and negative electrodes of the electromagnetic training device are imitated on both sides of the heart structure but are not in contact;
g)将多通道样品的血管系统与脉动生物反应器导流管相连，启动脉动培养系统，并控制其中培养液的流速为10mm/s，作用时间20min；g) connecting the vascular system of the multi-channel sample with the pulsating bioreactor guide tube, starting the pulsation culture system, and controlling the flow rate of the culture solution to be 10 mm/s, and the action time is 20 min;
h)将将电磁训练装置的电场发生装置通电，并控制电流大小5mA，直流；h) energizing the electric field generating device of the electromagnetic training device, and controlling the current size of 5 mA, DC;
i)完成电场对仿生肝肿瘤结构的训练后，将其继续进行体外培养。i) After completing the training of the electric field on the structure of the bionic liver tumor, it is continued to be cultured in vitro.
实施例11：利用多喷头3D打印法制备一种含血管、神经和免疫系统三通道的仿软骨结构，并将该仿软骨结构在电场中进行训练。Example 11: A multi-nozzle 3D printing method was used to prepare a pseudo-chondral structure containing three channels of blood vessels, nerves and immune system, and the pseudo-chondral structure was trained in an electric field.
a)利用计算机设计所述含多通道的仿软骨结构的三维模型； a) using a computer to design a three-dimensional model of the multi-channel-like pseudo-chondral structure;
b)将软骨细胞作为主体细胞，与配置好的纤维蛋白原溶液混合，纤维蛋白原溶液质量体积浓度为3％；之后将该混合物装载到多喷头3D打印设备的一个喷头组件中；b) the chondrocytes are used as host cells, mixed with the configured fibrinogen solution, and the fibrinogen solution has a mass volume concentration of 3%; the mixture is then loaded into a nozzle assembly of the multi-nozzle 3D printing device;
c)将血管内皮细胞、平滑肌细胞、神经元和T淋巴细胞作为通道细胞；将血管内皮细胞和平滑肌细胞与海藻酸钠溶液混合，装入3D打印设备的第二个喷头组件中；将神经元与海藻酸钠溶液混合，装入3D打印设备的第三个喷头组件中；将T淋巴细胞与海藻酸钠溶液混合，装入3D打印设备的第四个喷头组件中；c) using vascular endothelial cells, smooth muscle cells, neurons, and T lymphocytes as channel cells; mixing vascular endothelial cells and smooth muscle cells with sodium alginate solution, and loading them into a second nozzle assembly of the 3D printing device; Mixing with the sodium alginate solution, loading into the third nozzle assembly of the 3D printing device; mixing the T lymphocytes with the sodium alginate solution into the fourth nozzle assembly of the 3D printing device;
d)由计算机控制多喷头3D打印设备的不同喷头组件，控制主体结构的软骨细胞/纤维蛋白原的成形，并采用凝血酶使纤维蛋白原凝胶化；同步或异步控制毛细血管内皮细胞和平滑肌细胞/海藻酸钠的通道成形，得到血管系统通道；同步或异步控制神经元/海藻酸钠的通道成形，得到神经系统通道；同步或异步控制T淋巴细胞/海藻酸钠的通道成形，得到免疫系统通道；采用CaCl2使海藻酸钠溶液凝胶化得到海藻酸钠水凝胶，逐层堆积，得到所述含多通道的三通道的仿软骨结构。d) computer controlled multi-nozzle 3D printing device different nozzle components, control the formation of chondrocytes/fibrinogen of the main structure, and gelatinize fibrinogen by thrombin; control capillary endothelial cells and smooth muscle synchronously or asynchronously Channel formation of cells/sodium alginate to obtain vascular system channels; synchronous or asynchronous control of neuronal/sodium alginate channel formation to obtain neural system channels; simultaneous or asynchronous control of T lymphocyte/sodium alginate channel formation, immunization System channel; sodium alginate hydrogel was gelated by CaCl 2 to obtain sodium alginate hydrogel, and stacked layer by layer to obtain the multi-channel three-channel pseudo-chondral structure.
e)将制备好的仿软骨结构至于电磁训练装置的样品台上；e) preparing the prepared cartilage structure onto the sample stage of the electromagnetic training device;
f)将电磁训练装置的正极和负极的金属探针与仿软骨结构连接，使仿软骨结构成为电路一部分；f) connecting the metal probes of the positive and negative electrodes of the electromagnetic training device to the pseudo-chondral structure, so that the pseudo-chondral structure becomes a part of the circuit;
g)将将电磁训练装置的电场发生装置通电，并由控制电流大小为2mA，交流；g) the electric field generating device of the electromagnetic training device will be energized, and the control current is 2 mA, alternating current;
h)完成电场对仿软骨结构的训练后，将其继续进行体外培养。 h) After completing the training of the simulated cartilage structure by the electric field, it is continued to be cultured in vitro.

Claims (13)

  1. 一种含通道的仿生结构，其特征在于：所述仿生结构包括结构主体(101)和至少一个通道，所述通道分布在结构主体(101)内；所述结构主体为天然高分子水凝胶和细胞组成的混合物，所述细胞浓度为10-108个/mL，该细胞为胚胎干细胞、成体干细胞、成体细胞、癌细胞和诱导多能干细胞中的至少一种；所述通道为单通道或分支通道(105)，通道为两端通孔(103)、两端盲孔(106)和一端盲孔一端通孔结构(104)中的一种或几种组合；通道之间的位置关系为相交、平行、共线或异面；所述通道外壁、通道内壁或通道孔内分布有细胞，细胞为神经系统种子细胞、血管系统种子细胞和免疫系统种子细胞中的至少两种。A channel-containing biomimetic structure, characterized in that the biomimetic structure comprises a structural body (101) and at least one channel, the channel being distributed in the structural body (101); the structural body is a natural polymer hydrogel And a mixture of cells having a cell concentration of 10-10 8 /mL, the cell being at least one of an embryonic stem cell, an adult stem cell, an adult cell, a cancer cell, and an induced pluripotent stem cell; the channel is a single channel Or a branch channel (105), the channel is one or a combination of two ends of the through hole (103), the two ends of the blind hole (106), and one end of the blind hole one end through hole structure (104); the positional relationship between the channels It is intersecting, parallel, collinear or heterogeneous; cells are distributed in the outer wall of the channel, the inner wall of the channel or the pore of the channel, and the cells are at least two of the seed cells of the nervous system, the seed cells of the vascular system and the seed cells of the immune system.
  2. 如权利要求1所述的一种含通道的仿生结构，其特征在于：所述通道的截面为圆形、椭圆形、多边形或不规则几何图形，且通道截面面积为100μm2-1cm2。A channel-containing biomimetic structure according to claim 1, wherein the channel has a circular, elliptical, polygonal or irregular geometry and has a channel cross-sectional area of 100 μm 2 -1 cm 2 .
  3. 如权利要求1所述的一种含通道的仿生结构，其特征在于：所述神经系统种子细胞为神经元和神经胶质细胞中的至少一种；所述血管种子细胞为内皮细胞、平滑肌细胞和脂肪干细胞中的至少一种；所述免疫系统种子细胞为淋巴细胞和固有免疫细胞中的至少一种。The channel-containing biomimetic structure according to claim 1, wherein the seed cell of the nervous system is at least one of a neuron and a glial cell; and the blood vessel seed cell is an endothelial cell or a smooth muscle cell. And at least one of the adipose stem cells; the immune system seed cells being at least one of lymphocytes and innate immune cells.
  4. 如权利要求1所述的一种含通道的仿生结构，其特征在于：所述天然高分子水凝胶为海藻酸钠、胶原、基质胶、右旋糖、壳聚糖、明胶和纤维蛋白原中的至少一种，该水凝胶的质量体积浓度为0.1～20％。A channel-containing biomimetic structure according to claim 1, wherein said natural polymer hydrogel is sodium alginate, collagen, matrigel, dextrose, chitosan, gelatin and fibrinogen. At least one of the hydrogels has a mass to volume concentration of 0.1 to 20%.
  5. 如权利要求1所述的一种含通道的仿生结构，其特征在于：所述天然高分子水凝胶中复合有细胞冻存剂、细胞生长因子、药物、抗凝血剂和磁性纳米颗粒中的至少一种；所述细胞冻存剂为二甲基亚砜、甘油和右旋糖中的至少一种；所述细胞生长因子为血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子、人血小板衍化生长因子和转化生长因子中的至少一种；所述的药物为抗肿瘤药物和病毒疫苗中的至少一种；所述的抗凝血剂为肝素和紫杉醇中的至少一种；所述磁性纳米颗粒为铁氧体颗粒、金属型颗粒和氮化铁颗粒中的至少一种。A channel-containing biomimetic structure according to claim 1, wherein said natural polymer hydrogel is compounded with cell cryopreservation agent, cell growth factor, drug, anticoagulant and magnetic nanoparticle. At least one of the cell cryopreservatives is at least one of dimethyl sulfoxide, glycerol and dextrose; the cell growth factor is vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte At least one of a growth factor, a human platelet-derived growth factor, and a transforming growth factor; the drug is at least one of an antitumor drug and a viral vaccine; the anticoagulant is at least one of heparin and paclitaxel The magnetic nanoparticles are at least one of ferrite particles, metal particles, and iron nitride particles.
  6. 一种含通道的仿生结构的电磁力训练装置，其特征在于：所述装置包括电场发生系统(601)、磁场发生系统、样品台(603)和固定平台(605)；所述磁场发生系统和样品台(603)安装于固定平台(605)上；所述电场发生系(601)统含正极和负极；所述样品台(603)设有导轨，所述正极和负极分别通过滑块安装在导轨上；所述样品台(603)位于磁场发生系统内；所述磁场发生系统分包括支架(602)和可旋转圆环(608)，所述可旋转圆环(608)安装在支架(602)的中空结构内，在可旋转圆环(608)上径向对称排布有可拆卸的S极(606)和N极(607)。An electromagnetic force training device with a channel-like bionic structure, characterized in that the device comprises an electric field generating system (601), a magnetic field generating system, a sample stage (603) and a fixed platform (605); the magnetic field generating system and The sample stage (603) is mounted on the fixed platform (605); the electric field generating system (601) comprises a positive electrode and a negative electrode; the sample stage (603) is provided with a guide rail, and the positive electrode and the negative electrode are respectively mounted on the slider The sample stage (603) is located within the magnetic field generating system; the magnetic field generating system includes a bracket (602) and a rotatable ring (608), and the rotatable ring (608) is mounted on the bracket (602) In the hollow structure, a detachable S pole (606) and an N pole (607) are radially symmetrically arranged on the rotatable ring (608).
  7. 如权利要求6所述的一种含通道的仿生结构的电磁力训练装置，其特征在于：所述装置还包括脉动培养系统(701)；所述脉动培养系统安装于固定平台(605)上；所述脉动培养系统(701)包括脉动系统电机(702)、脉动系统导轨-滑块机构(703)、培养液供给注射器(704)、单向阀(705)、导流管(706)和培养液瓶(708)；所述脉动系统电机(702)通过曲柄与脉动系统导轨-滑块机构(703)连接；所述脉动系统导轨-滑块机构(703)与培养液供给注射器(704)连接。 The electromagnetic force training device for a channel-containing bionic structure according to claim 6, wherein the device further comprises a pulsation culture system (701); the pulsation culture system is mounted on the fixed platform (605); The pulsation culture system (701) includes a pulsation system motor (702), a pulsation system guide-slider mechanism (703), a culture fluid supply syringe (704), a check valve (705), a draft tube (706), and a culture a liquid bottle (708); the pulsation system motor (702) is coupled to a pulsating system rail-slider mechanism (703) by a crank; the pulsating system rail-slider mechanism (703) is coupled to a culture fluid supply injector (704) .
  8. 如权利要求6或7所述的一种含通道的仿生结构的电磁力训练装置，其特征在于：所述正极和负极为金属平板、金属导线或金属探针中的一种。The electromagnetic force training device for a channel-containing bionic structure according to claim 6 or 7, wherein the positive electrode and the negative electrode are one of a metal plate, a metal wire or a metal probe.
  9. 如权利要求6或7所述的一种含通道的仿生结构的电磁力训练装置，其特征在于：所述S极和N极为永磁体或电磁铁中的一种。The electromagnetic force training device for a channel-containing bionic structure according to claim 6 or 7, wherein the S pole and the N are one of a permanent magnet or an electromagnet.
  10. 一种采用如权利要求6所述装置对含通道的仿生结构进行训练的方法，其特征在于，所述方法包括如下步骤：A method of training a channel-containing bionic structure using the apparatus of claim 6 wherein the method comprises the steps of:
    a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
    b)启动电磁训练装置的电场发生系统，移除磁场发生系统的N级和S级；使含通道的仿生结构位于电场中；电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；b) starting the electric field generating system of the electromagnetic training device, removing the N and S stages of the magnetic field generating system; and making the bionic structure containing the channel in the electric field; the current is greater than 0, less than or equal to 50 mA; the voltage is greater than 0, less than or equal to 50 V The current direction is alternating between AC, DC or both;
    c)完成电场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the training of the electric field on the bionic structure containing the channel, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  11. 一种采用如权利要求6所述装置对含通道的仿生结构进行训练的方法，其特征在于，所述方法包括如下步骤：A method of training a channel-containing bionic structure using the apparatus of claim 6 wherein the method comprises the steps of:
    a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
    b)关闭电磁训练装置的电场发生系统，启动磁场发生系统，使S极和N极随可旋转圆环旋转，控制磁感应强度为大于0，小于等于5T；b) turning off the electric field generating system of the electromagnetic training device, starting the magnetic field generating system, so that the S pole and the N pole rotate with the rotatable ring, and the control magnetic induction intensity is greater than 0, less than or equal to 5T;
    c)完成磁场对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the training of the magnetic field on the channel-containing bionic structure, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  12. 一种采用如权利要求6所述装置对含通道的仿生结构进行训练的方法，其特征在于，所述方法包括如下步骤：A method of training a channel-containing bionic structure using the apparatus of claim 6 wherein the method comprises the steps of:
    a)将制备好的含通道的仿生结构浸入含悬浮细胞的细胞培养液中，并置于电磁力训练装置的样品台上；a) immersing the prepared channel-containing biomimetic structure in a cell culture medium containing suspended cells, and placing it on a sample stage of the electromagnetic force training device;
    b)同时启动电磁训练装置的电场发生系统和磁场发生系统，使含通道的仿生结构位于电场和磁场中；控制电流为大于0，小于等于50mA；电压为大于0，小于等于50V；电流方向采用交流、直流或两者交替使用；控制磁感应强度为大于0，小于等于5T；b) simultaneously start the electric field generating system and the magnetic field generating system of the electromagnetic training device, so that the bionic structure containing the channel is located in the electric field and the magnetic field; the control current is greater than 0, less than or equal to 50 mA; the voltage is greater than 0, less than or equal to 50 V; AC, DC or both are used alternately; the control magnetic induction intensity is greater than 0, less than or equal to 5T;
    c)完成电场和磁场同时对含通道的仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。c) After completing the electric field and magnetic field training of the channel-containing bionic structure, the bionic structure is cryopreserved or continuously cultured in vitro or directly used for organ transplantation.
  13. 根据权利要求10、11或12所述的一种对含通道的仿生结构进行训练的方法，其特征在于：启动脉动培养系统(701)，所述脉动培养系统(701)包括脉动系统电机(702)、脉动系统导轨-滑块机构(703)、培养液供给注射器(704)、单向阀(705)、导流管(706)和培养液瓶(708)；所述脉动系统电机(702)通过曲柄与脉动系统导轨-滑块机构(703)连接；所述导轨-滑块机构与 培养液供给注射器(704)连接；通过导流管(706)将所述仿生结构与脉动培养系统连接；使培养液在导流管(706)和含通道的仿生结构之间进行单向流动；液体流速为大于0，小于30cm/s，进行脉动-电场、脉动-磁场或脉动-电磁复合场的作用；完成对所述仿生结构的训练后，将该仿生结构低温保存，或继续进行体外培养或直接用于器官移植。 A method of training a channel-containing bionic structure according to claim 10, 11 or 12, characterized in that a pulsation culture system (701) is activated, said pulsation culture system (701) comprising a pulsation system motor (702) a pulsating system rail-slider mechanism (703), a culture fluid supply syringe (704), a check valve (705), a draft tube (706), and a culture fluid bottle (708); the pulsation system motor (702) Connected to the pulsating system rail-slider mechanism (703) by a crank; the rail-slider mechanism and The culture fluid supply syringe (704) is connected; the biomimetic structure is connected to the pulsation culture system through a draft tube (706); and the culture fluid is unidirectionally flowed between the draft tube (706) and the channel-containing biomimetic structure; The liquid flow rate is greater than 0 and less than 30 cm/s, and the pulsation-electric field, the pulsation-magnetic field or the pulsation-electromagnetic composite field is performed; after the training of the bionic structure is completed, the bionic structure is cryopreserved or the in vitro culture is continued. Or directly used for organ transplantation.
PCT/CN2014/089771 2014-10-11 2014-10-29 Bionic structure containing channels and electromagnetic force training device and method therefor WO2016054847A1 (en)

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