CN113528337A - Combined die for organ manufacturing and drug screening and use method thereof - Google Patents

Abstract

The invention belongs to the technical field of organ manufacturing and drug screening, and particularly relates to a combined die for organ manufacturing and drug screening and a using method thereof. The method comprises the following steps: the device comprises a base die, an outer layer orifice die, an inner layer orifice die, five vertical channel dies and a horizontal channel die. The outer layer orifice plate die is arranged outside the inner layer orifice plate die to form a two-layer structure, a gap is reserved between the two layers, and the two layers are embedded in the same base die; the vertical channel die is positioned in the inner-layer pore plate die and embedded into the groove of the base die. The outer walls of the outer layer pore plate die and the inner layer pore plate die are both provided with a plurality of embedding holes, and the horizontal channel die is inserted into the embedding holes; the molds are combined to simulate a multi-channel complex organ. Adopts the bionics principle to imitate various complex structures in organs, overcomes various defects of constructing complex organs containing various cells and screening drugs in the prior art, and realizes the manufacture of complex organs containing multiple channels and high-flux drug screening.

Description

Combined die for organ manufacturing and drug screening and use method thereof

Technical Field

The invention belongs to the technical field of organ manufacturing and drug screening, and particularly relates to a combined die for organ manufacturing and drug screening and a using method thereof.

Background

Tissue engineering is an emerging interdiscipline that integrates the principles and methods of life science, material science, and engineering science and aims to recreate human tissues and organs. In recent years, the tissue engineering research has made breakthrough progress in the reconstruction of skin, cartilage, nerve, blood vessel and myocardial tissues, and some tissue engineering products enter clinical application. Tubular networks such as blood vessels and nerve conduits are abundant in internal organs. The blood vessel network provides oxygen and nutrition for each part of tissue cells of the organ and simultaneously discharges metabolic waste out of the body, thereby ensuring the normal operation of various physiological functions. The lack of a sufficiently fine vascular network makes it difficult to construct tissue of large volume. At present, the construction of vascular and nerve conduit networks has become a major research hotspot in the field of biological artificial organ manufacturing. The commonly used method for constructing the vascular and nerve conduit network mainly comprises the steps of slowly promoting the formation of different tissue layers of the blood vessel by using corresponding growth factors, forming a vascular structure by multi-cell combined culture, forming the blood vessel by using technologies such as a bioreactor, a microfluidic channel and the like, directly constructing the vascularized tissue based on an advanced forming technology and the like.

One of the reasons that the price of drugs cannot be reduced at present is the lack of an experimental model system that can replace expensive and time-consuming animal studies. Although cell culture models have made considerable progress as tissue and organ substitutes in these types of studies, cultured cells are often unable to maintain differentiation and expression of tissue-specific functions. Although extracellular matrix (ECM) gels can promote cell growth, the mechanical properties of organs constructed using such hydrogels are inadequate and the three-dimensional structural complexity is limited. In particular, existing organ construction models fail to reconstruct an active tissue-tissue interface between the microvascular endothelium and the adjacent parenchymal tissues where critical transport of fluids, nutrients, immune cells and other regulatory factors occurs, and it is difficult to apply dynamic mechanical forces (e.g., respiratory motion of the lungs, shearing of blood vessels, peristalsis of the intestinal tract, skin tension, etc.) that are critical to the development and function of living organs. The use of multi-microtubule channels makes possible the integrated or integrated construction of various complex structures in organ manufacture, including blood vessels, muscles, bones, respiratory tract, liver, brain, intestinal tract, and kidneys. For example, the introduction of a tracheal system (or network) in the lung architecture, which introduces air into the epithelial compartment as lung parenchyma cells grow to confluence, creates an air-liquid interface that more accurately mimics the lining of the alveolar air space, and wherein the compartmentalized channel structure controls the flow of fluid and the independent delivery of cells and nutrients to the epithelium and endothelium.

The invention patent (application No. 201110448154.3) discloses a method for preparing a complex organ precursor with multi-branch and multi-layer structure based on a combined mold, which utilizes the combined mold to perform layered perfusion to form the complex organ precursor with multi-branch structure. The invention has the disadvantages that the direct demoulding mode is adopted, the formed cell matrix layer can be damaged by the solid multi-branch inner mould due to the irregular shape during demoulding, and the preparation method has higher requirements on the internal shape and the roughness of the mould. Meanwhile, as the contact surface of the cross-linking agent or the polymerizing agent and the high molecular solution material is too small, the cross-linking or polymerizing effect can be easily influenced, and the forming effect of a cell matrix layer is further influenced. Since the control of the cell microenvironment with high precision is not possible, it is not suitable for drug screening.

The invention patent (application No. 201210324600.4) discloses a method for preparing a spindle-shaped complex organ precursor by using a rotary combined mold. In theory, a complicated organ in the form of a spindle with a multi-branched tubular passage can be formed by the rotation of the mold, but the disadvantage is that the rotational speed of the mold is different, the molding height of the spindle is different, and the rotational speed is difficult to be quantitatively limited for different combined molds, so that it is difficult to determine the specific molding shape of the spindle. The combined mould is only used for forming the spindle organ precursor containing a group of horizontal channels at present, and is easy to cause the damage of the structure of the material containing cell matrix when the inner mould is pulled out, and the cell activity is influenced by rotating.

The invention patent (application No. 201510419730. X) discloses a method and a special mold for preparing a tissue organ precursor with multiple horizontal channels, which proposes a special mold for preparing the tissue organ precursor with multiple horizontal channels, and has the defects of complicated intermediate operation steps and easy dislocation among large and small pipelines. The hydrogel solution with slightly high viscosity is difficult to fill the whole inner cavity, the branch inner mold is not detachable, and the formed structure can be damaged when the hydrogel solution is taken out from the cell matrix layer.

The invention patent (application No. 201910682212.5) discloses a detachable special mold and a method for preparing a multi-horizontal-channel complex organ precursor. The branch mold in the special mold can be detached, and the formed structure cannot be damaged when the branch mold is taken out from the cell matrix layer.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a combined die for organ manufacturing and drug screening and a using method thereof. The bionic method is used for simulating a complex organ structure by utilizing a bionic principle and designing structures such as a vertical channel mold, a horizontal channel mold, an orifice plate mold and the like to obtain the complex organ structure with a large channel in the middle and a plurality of small channels around and different cell matrix layers. In particular to an in vitro manufacturing and drug screening method for blood vessels, nerve conduits, trachea and bronchus with multi-branch channels.

In order to achieve the above object, the present invention adopts the following technical solution for a combined mold for organ manufacture and drug screening, comprising: the device comprises a base die, an outer layer orifice die, an inner layer orifice die, five vertical channel dies and a horizontal channel die.

The outer layer orifice plate die is arranged outside the inner layer orifice plate die to form a two-layer structure, a gap is reserved between the two layers, and the two layers are embedded in the same base die; the vertical channel die is positioned in the inner-layer pore plate die and embedded into the groove of the base die.

The outer walls of the outer layer pore plate die and the inner layer pore plate die are both provided with a plurality of embedding holes, and the horizontal channel die is inserted into the embedding holes; the molds are combined to simulate a multi-channel complex organ.

Further, the base mould is rectangular, the bottom surface is a plane or a concave surface or a convex surface, a cylindrical groove used for fixing the vertical channel mould is arranged in the middle of the base mould, four rectangular grooves used for fixing four outer-layer orifice plate moulds are respectively arranged on four sides of the rectangular base mould, and four grooves used for fixing the inner-layer orifice plate mould are further arranged in the base mould.

The outer layer hole plate die comprises an outer layer hole plate die I, an outer layer hole plate die II, an outer layer hole plate die III and an outer layer hole plate die IV; the four outer layer pore plate moulds are embedded into the rectangular grooves of the corresponding base moulds.

The inner-layer pore plate die comprises a first inner-layer pore plate die, a second inner-layer pore plate die, a third inner-layer pore plate die and a fourth inner-layer pore plate die; and the four inner-layer pore plate molds are embedded into the corresponding grooves of the base mold.

The horizontal channel molds are divided into two groups, each group has three layers, the two groups of channels are crossed into 90 degrees, and each layer has six branches. The two groups of horizontal channels are fixed by inserting the corresponding embedding holes in the pore plate die, and the length of the horizontal channel die is greater than the distance between the two corresponding outer layer pore plate dies.

Further, the complex organ is a complex organ structure with a middle large channel, a plurality of small channels and different cell matrix layers; the complex organ is a complex organ with multi-branched channels of blood vessels, nerve conduits, trachea and bronchi.

The five vertical channels are used for simulating thicker pipelines in organs, wherein the thicker pipelines comprise a trachea in a lung, a pancreatic duct in a pancreas and a bile duct in a liver; and a plurality of vertical channels can ensure that nutrient substances enter and exit at one time during in-vitro culture, thereby realizing the pulse culture of nutrient substance circulation.

The vertical channel mould adopts a spindle-shaped branch structure, and holes are punched on the pipe wall; the contact area between the nutrient substances is increased, the nutrient substances are absorbed conveniently, the metabolic waste is transported conveniently, and the drug screening is more convenient.

Multiple horizontal channel molds are used to mimic the finer channels in an organ, including small vessels surrounding the middle arteriovenous vessels, nerve conduits, bronchial structures (pulsatile culture is possible because both vertical and horizontal channels are through structures).

Furthermore, the two-layer structure is made of hydrogel materials and used for culturing specific tissues at different parts of an organ, and different pipelines can be used for culturing different cells.

Still further, the different cells include vascular epithelial cells, nerve, bronchial epithelial cells.

Furthermore, the base mold, the five vertical channel molds, the outer layer orifice plate mold, the horizontal channel mold and the inner layer orifice plate mold are all made of synthetic polymer materials (such as synthetic fibers, polyethylene, polycarbonate, polyvinyl chloride, photosensitive tree branches or polyurethane) or metal materials without biotoxicity.

Further, the diameter of the horizontal channel die is 0.1-5 mm; the diameter of the vertical channel die is the same as that of the corresponding groove on the base die; the groove of the base mold is the same as the thickness of the corresponding floor slab mold (to ensure good sealing).

A method of using a combined mold for organ manufacture and drug screening, comprising the steps of:

step 1, (extraction or purchase) preparation of a cell suspension from animal or human cells, the cell suspension having a density of 1X 10³～5×10⁷Preparing 1-30% of natural polymer hydrogel solution serving as a matrix solution, and mixing the matrix solution and the cell suspension according to a volume ratio of 1-9: 9-1, and mixing to obtain a matrix solution containing cells, wherein a plurality of matrix solutions containing one (or more different) cells can be obtained by changing the selected cell type and the type of the matrix solution.

And 2, coating a layer of matrix solution containing cells (such as tracheal endothelial cells, vascular endothelial cells and the like) on all the channel molds, and obtaining a layer of tubular cell matrix layer by adopting a physical and chemical crosslinking or polymerization method, wherein the cell matrix layer on the surface of each tubular channel needs to have a certain thickness.

And 3, assembling all components of the combined die: firstly, fixing an inner layer of pore plate mould and an outer layer of pore plate mould, then inserting the horizontal channel mould which is subjected to cross-linking treatment in the step 2 into corresponding holes on the pore plate mould, and fixing five vertical channel moulds in grooves of a base mould; adding a matrix solution containing cells into the inner cavity of the mold consisting of the inner-layer pore plate to fully fill the whole inner cavity with the matrix solution; preparing an inner cell matrix layer by using a physical and chemical crosslinking or polymerization method; then the horizontal channel mould is withdrawn to the outer layer hole plate mould, the inner layer hole plate mould is taken out, and the horizontal channel mould is restored to the original position; then another matrix solution containing cells is added into the whole inner cavity of the outer layer consisting of the outer layer pore plate mould and the inner layer pore plate mould so that the matrix solution fully fills the whole inner cavity. The outer cell matrix layer is made using physical, chemical cross-linking or polymerization methods.

And 4, pulling out all the installed horizontal channel molds by using forceps, and forming the artificial organ containing the vascular endothelial cells, or (and) bronchial mucosal endothelial cells, or (and) nerve conduit endothelial cells by adding the seed cells, the culture medium and the growth factors and performing targeted culture.

And 5, dissolving the medicine in a cell culture solution or a PBS solution, injecting the medicine into a pipeline on the combined die, allowing the medicine to flow through the artificial organ containing the multicellular layer, regularly extracting the liquid in the pipeline, measuring the concentration change of the medicine, characterizing the cell shape, and identifying the reaction degree of the cell to the medicine.

In the first preferred scheme, the hydrogel solution is one or more of collagen, agarose, fibrinogen, sodium alginate, silk fibroin, gelatin, hyaluronic acid, chitosan, fibronectin, thrombin and polyethylene glycol (the hydrogel solution obtained by mixing a plurality of materials has a good effect).

In the second preferred scheme, the combined die can form multiple cell matrix layers containing different cells to complete the assembly of various cell tissues.

Adding cell growth factors and anticoagulant factors into the cell matrix solution, wherein the volume percentage of the cell growth factors and the anticoagulant factors in the total solution is 0.001-0.1%; the cell growth factor comprises one or more of endothelial cell growth factor, cell transfer factor or lung cell growth factor, and the anticoagulant factor comprises one or more of heparin, paclitaxel or sulfated chitosan.

Compared with the prior art, the invention has the beneficial effects.

The invention adopts the bionics principle to imitate various complex structures in organs, overcomes various defects of constructing complex organs containing various cells and screening medicines in the prior art, and realizes the manufacture of complex organs containing multiple channels and high-throughput medicine screening.

Drawings

The invention is further described with reference to the following figures and detailed description. The scope of the invention is not limited to the following expressions.

Fig. 1 is an overall sectional view of a split mold.

Fig. 2 is an overall sectional exploded view of the split mold.

FIG. 3 is a top view of a split mold.

Fig. 4 is a left side view of the split mold.

Fig. 5 is a perspective view of a split mold.

Fig. 6 is a multi-empty vertical channel with spindle-shaped branches.

FIG. 7 is a graphical representation of a complex organ precursor matrix with a multi-channel network (organ precursors with three different cell matrix layers are used as an example).

Fig. 8 is an exploded view of the combined mold.

In the figure, 201-first cell matrix layer; 202-second cell matrix layer.

Detailed Description

The invention relates to a three-dimensional structure artificial manufacturing and drug screening technology for a complex organ containing multiple channels, in particular to a special combined die for constructing the complex organ containing multiple branch channels by adopting a bionics principle, and a process method for manufacturing the complex organ and screening drugs by utilizing the die and high polymer materials, cells, growth factors and the like, which exceed the technical range of the existing tissue engineering, organ manufacturing and drug screening.

As shown in fig. 1-8, the present invention includes a base mold 121, a first vertical channel mold 103, a second vertical channel mold 105, a third vertical channel mold 106, a fourth vertical channel mold 107, and a fifth vertical channel mold 108.

The outer layer hole plate die I101, the outer layer hole plate die II 109, the outer layer hole plate die III 111 and the outer layer hole plate die IV 114. The first inner-layer orifice plate die 102, the second inner-layer orifice plate die 110, the third inner-layer orifice plate die 112, the fourth inner-layer orifice plate die 113, the first horizontal channel die 115, the second horizontal channel die 116, the third horizontal channel die 117, the fourth horizontal channel die 118, the fifth horizontal channel die 119 and the sixth horizontal channel die 120.

The base mold 121 is rectangular, the bottom surface is a plane, a concave surface or a convex surface, a cylindrical groove capable of fixing the vertical channel mold is arranged in the middle, rectangular grooves for fixing four outer-layer orifice plate molds are respectively arranged on the periphery of the outermost edge, and four rectangular grooves for fixing inner-layer orifice plate molds are inwards arranged.

The horizontal channel mould is total two sets of, and every group has the three-layer, and two sets of passageways intersect into 90 degrees, and each layer all has six branches, and two sets of horizontal channels are fixed through the aperture that corresponds on the patchhole board mould, and horizontal channel mould length is greater than the distance between two opposite outer layer hole board moulds, convenient to detach, can carry out the independent assortment according to specific needs moreover.

The vertical channel adopts a spindle-shaped branch structure and holes are formed in the tube wall, so that the contact area between the vertical channel and nutrient substances is increased, the nutrient substances are convenient to absorb and the metabolic waste is convenient to transport, and the drug screening is more convenient.

Preferably, the combined mould simulates the structure of a specific organ by adopting a bionic method, and the five vertical channels fixed at the center of the base mould can simulate thicker pipelines in the organ, such as a trachea in a lung, a pancreatic duct in a pancreas, a bile duct in a liver and the like. And during in vitro culture, the vertical channels can ensure that nutrient substances enter and exit at one time, so that the pulse culture of nutrient substance circulation is realized, the horizontal channel molds can simulate thin pipelines in organs, such as small vessels surrounding middle arteriovenous vessels, nerve conduits and bronchial structures, and the pulse culture can be realized because the vertical channels and the horizontal channels are communicated structures.

Preferably, the combined die adopts a structure of two layers of orifice plates from inside to outside. The inner-to-outer layer structure can select the most suitable hydrogel material for culturing specific tissues at different parts of an organ, and different pipelines can be used for culturing different cells, such as vascular epithelial cells, nerve and bronchial epithelial cells and the like.

Preferably, the base mold, the five vertical channel molds, the frame mold, the horizontal channel mold and the orifice mold are made of synthetic polymer materials (such as synthetic fibers, polyethylene, polycarbonate, polyvinyl chloride, photosensitive tree branches or polyurethane) or metal materials without biotoxicity.

Preferably, the diameter of the horizontal channel die ranges from 0.1mm to 5 mm; the grooves reserved on the diameter base mold of the vertical channel mold 138 are the same; the grooves of the base mold 143 correspond to the same thickness as the orifice plate mold to ensure good sealing.

The combined mould and the use method for organ manufacturing and drug screening have the following specific process steps.

(1) Extracting or purchasing animal or human cells to obtain cell suspension with density of 1 × 10³～5×10⁷Preparing 1-30% of natural polymer hydrogel solution serving as a matrix solution, and mixing the matrix solution and the cell suspension according to a volume ratio of 1-9: 9-1, and mixing to obtain a matrix solution containing cells, wherein a plurality of matrix solutions containing one (or more different) cells can be obtained by changing the selected cell type and the type of the matrix solution.

(2) Coating a layer of matrix solution containing cells (such as tracheal endothelial cells, vascular endothelial cells, etc.) on all channel molds, and crosslinking by using a physical and chemical crosslinking or polymerization method to obtain a layer of tubular cell matrix layer, wherein the cell matrix layer on the surface of each tubular channel has a certain thickness.

(3) Assembling the components of the combined die: firstly, fixing an inner layer orifice plate mould and an outer layer orifice plate mould, then inserting the horizontal channel mould which is subjected to cross-linking treatment in the step (2) into corresponding holes on the orifice plate mould, and then fixing five vertical channel moulds in the grooves of the base mould. A matrix solution containing cells is added to the cavity of the mold consisting of the inner-layer well plate so that the matrix solution substantially fills the entire cavity. The inner cell matrix layer is made using physical, chemical cross-linking or polymerization methods. And then withdrawing the horizontal channel mould to the outer layer hole plate mould, taking out the inner layer hole plate mould, and restoring the horizontal channel mould to the original position. Then another matrix solution containing cells is added into the whole inner cavity of the outer layer consisting of the outer layer pore plate mould and the inner layer pore plate mould so that the matrix solution fully fills the whole inner cavity. The outer cell matrix layer is made using physical, chemical cross-linking or polymerization methods.

(4) All the installed horizontal channel molds are pulled out by using forceps, and the artificial organs containing vascular endothelial cells, or (and) bronchial mucosal endothelial cells, or (and) nerve conduit endothelial cells are pertinently cultured by adding seed cells, culture media and growth factors.

(5) Dissolving the medicine in cell culture solution or PBS solution, injecting the solution into a pipeline on a combined die, allowing the solution to flow through an artificial organ containing a multicellular layer, regularly extracting the liquid in the pipeline, measuring the concentration change of the medicine, characterizing the cell shape, and identifying the reaction degree of the cell to the medicine.

The preferable scheme of the invention is that the natural polymer material of the prepared hydrogel solution usually adopts one or more of materials such as collagen, agarose, fibrinogen, sodium alginate, silk fibroin, gelatin, hyaluronic acid, chitosan, fibronectin, thrombin, polyethylene glycol and the like, and the hydrogel solution obtained by mixing a plurality of materials has better effect. The solvent used for dissolving the natural polymer material in the extraction part is water, a PBS solution, normal saline, a 3-hydroxymethyl aminomethane hydrochloric acid solution, 0.09M sodium chloride with the pH value of 6-8 or a cell culture solution. One or more of cell growth factors such as endothelial cell growth factor, cell transfer factor or lung cell growth factor and one or more of anticoagulant factors such as heparin, paclitaxel or sulfated chitosan can be added into the cell matrix solution, and the volume percentage of the cell growth factors and the anticoagulant factors in the total solution is 0.001-0.1%.

The base mold, the two vertical channel molds, the horizontal channel mold and the orifice plate mold are made of synthetic high polymer materials (such as synthetic fibers, polyethylene, polycarbonate, polyvinyl chloride, photosensitive tree branches or polyurethane) or metal materials without biotoxicity.

The diameter range of the horizontal channel die is 0.1-5 mm; the grooves reserved on the diameter base mold of the vertical channel mold 138 are the same; the rectangular groove of the base mold 143 corresponds the same thickness as the orifice mold to ensure good sealing.

Specific example 1: lung manufacture with multiple branch vascular networks and drug screening.

(1) A base mold, five vertical channel molds, a horizontal channel mold, and an orifice plate mold were prepared using a photosensitive resin.

(2) A1% fibrinogen solution and a thrombin solution (20 IU/ml) were prepared.

(3) All channel molds were coated with a mixture of endothelial cells and fibrinogen, the matrix solution having a cell density of 5X 10⁷Per ml, 1% heparin was added to the matrix solution in weight/volume (w/v), and fibrinogen was polymerized for 2 min using thrombin solution (20 IU/ml) to obtain a tubular cell matrix layer.

(4) Assembling the components of the combined die: fixing the orifice plate die and the five vertical channel dies on the base die through the grooves, and then inserting all the horizontal channel dies in the step (3) into corresponding holes of the orifice plate die. Cell density 5X 10 through the top⁷A mixed matrix solution of lung tissue cells and fibrinogen per ml is added into the inner cavity formed by the inner-layer pore plate mould, so that the matrix solution fully fills the whole mould inner cavity. Fibrinogen was polymerized using thrombin solution (20 IU/ml) to form the inner cell matrix layer. And then the horizontal channel die is withdrawn to the outer layer orifice die, and the inner layer orifice die is taken out.Finally, the cells were packed through the top at a density of 2X 10⁵Adding a mixed matrix solution of lung tissue cells and fibrinogen per ml into the inner cavity of the mold to fully fill the inner cavity consisting of the outer layer orifice plate mold and the inner layer orifice plate mold with the matrix solution. Fibrinogen was polymerized using thrombin solution (20 IU/ml) to form the bottom cell matrix layer.

(5) Pulling out all the installed horizontal channel molds with tweezers, and adding matrix solution mixed with lung tissue cells and fibrinogen into the channel through the formed horizontal channel opening, wherein the cell density is 2 x 10⁵And (4) polymerizing fibrinogen by using a thrombin solution (20 IU/ml) to prepare a matrix layer suitable for the growth of the duct cells, namely the biological artificial lung.

(6) Dissolving the anti-lung cancer drug in cell culture solution or PBS solution, injecting the solution into a pipeline on a combined die, allowing the solution to flow through the artificial lung containing the multicellular layer, regularly extracting the liquid in the pipeline, measuring the concentration change of the drug in the liquid, characterizing the cell shape in the liquid, and identifying the reaction degree of the cell to the drug.

Specific example 2: liver manufacture with multiple branch vessels and bile duct network and drug screening.

(1) A base mold, five vertical channel molds, a horizontal channel mold and a pore plate mold are prepared by using polytetrafluoroethylene.

(2) Mixing 5% gelatin and 2% sodium alginate, and dissolving in PBS solution as gelatin/sodium alginate matrix solution, wherein endothelial cell growth factor (EGFs 0.5 ng/ml) is added. A1% (w/v) calcium chloride solution was prepared.

(3) All channel molds were coated with a layer of adipose-containing stem cells (1X 10)⁵One/ml) and endothelial cell growth factor (EGFs 0.5 ng/ml), cross-linking the sodium alginate with 1% (w/v) calcium chloride solution to obtain a tubular inner matched cell layer.

(4) Assembling the components of the combined die: firstly, fixing the orifice plate mould and five vertical channel moulds on a base mould through grooves, and then inserting all the horizontal channel moulds in the step (3) into the orifice plate mould to correspond to the orifice plate mouldIn the hole of (a). Cell density 3X 10 through the top⁶Adding the hepatocyte and gelatin/sodium alginate matrix solution into the inner cavity formed by the inner layer pore plate mould to fully fill the matrix solution into the whole mould inner cavity. The inner cell matrix layer was made by cross-linking sodium alginate with 1% (w/v) calcium chloride solution. And then taking out the inner layer orifice plate mould. Then the cell density is 1X 10³Adding bile duct epithelial cells and gelatin/sodium alginate matrix solution into the inner cavity formed by the inner layer frame mould and the outer layer pore plate mould, and fully filling the whole inner cavity with the matrix solution. The sodium alginate was cross-linked using 1% (w/v) calcium chloride solution to make the outer cell matrix layer.

(5) Pulling out all the installed horizontal channel molds and vertical channel molds with tweezers, and adding adipose-derived stem cells (1X 10) into the channels through the formed channel openings⁵One/ml) and endothelial cell growth factor (EGFs 0.5 ng/ml), cross-linking sodium alginate with 1% (w/v) calcium chloride solution to obtain a middle layer cell matrix layer.

(6) Dissolving the drug in cell culture solution or PBS solution, injecting the solution into an artificial liver which flows through a multi-cell layer in a pipeline on a combined die, extracting the liquid in the pipeline at regular time, measuring the concentration change of the drug, characterizing the cell shape, and identifying the reaction degree of the cell to the drug.

Specific example 3: kidney manufacturing with multiple branch vessels, nerve conduit networks and drug screening.

(1) The base mold, five vertical channel molds, a horizontal channel mold, and an orifice mold were prepared using silicone rubber.

(2) A10% gelatin solution was prepared as a base solution and a 10% (w/v) transglutaminase (mTG, 100U/ml) solution.

(3) Coating a layer of mixture of Schwann cells and matrix solution on one half of the channel mold, and coating a layer of mixture of vascular epithelial cells and matrix solution on the other half of the channel mold, wherein the density of the cells in the matrix solution is 5 × 10⁶Each ml is cross-linked with transglutaminase, and left at 37 deg.C for 10 minSo that the mixture of gelatin and cells has stable structure to obtain a layer of tubular cell matrix layer, and the cell matrix layer on the surface of each tubular channel has a certain thickness.

(4) Assembling each component of the special combined die: firstly, fixing the orifice plate die and the five vertical channel dies on the base die through the grooves, and then inserting (3) all the horizontal channel dies into corresponding holes of the orifice plate die. Cell density 1X 10 through the top³A volume/ml of glomerular cells and gelatin solution is added to the cavity formed by the inner orifice plate mold so that the matrix solution substantially fills the entire mold cavity. The bottom cell matrix layer is made by using transglutaminase for cross-linking. And then the horizontal channel die is withdrawn to the outer layer orifice die, and the inner layer orifice die is taken out. The cells were then passed overhead at a density of 1X 10³And adding the adipose-derived stem cells and the gelatin solution into the inner cavity of the mold in a per ml mode to ensure that the matrix solution fully fills the inner cavity consisting of the outer layer orifice plate mold and the inner layer orifice plate mold. The outer cell matrix layer is prepared by using transglutaminase for crosslinking.

(5) Pulling out all the installed horizontal channel molds with tweezers, and adding adipose-derived stem cells and gelatin solution into the channels through the formed channel openings, wherein the cell density is 1 × 10⁶Adding nerve cell growth factor (IGFs 0.1 ng/ml) into the matrix solution, and crosslinking with transglutaminase to obtain inner and outer cell matrix layers.

(6) Dissolving the drug in cell culture solution or PBS solution, injecting the solution into a pipeline on a combined die, allowing the solution to flow through the artificial kidney containing the multicellular layer, extracting the liquid in the pipeline at regular time, measuring the concentration change of the drug in the liquid, characterizing the cell shape in the liquid, and identifying the reaction degree of the cell to the drug.

It should be understood that the detailed description of the present invention is only for illustrating the present invention and is not limited by the technical solutions described in the embodiments of the present invention, and those skilled in the art should understand that the present invention can be modified or substituted equally to achieve the same technical effects; as long as the use requirements are met, the method is within the protection scope of the invention.

Claims (10)

1. A combined die for organ manufacture and drug screening comprises a base die, an outer layer orifice plate die, an inner layer orifice plate die, five vertical channel dies and a horizontal channel die;

the outer-layer pore plate die is arranged outside the inner-layer pore plate die to form a two-layer structure, a gap is reserved between the two layers, and the two layers are embedded in the same base die; the vertical channel die is positioned in the inner-layer pore plate die and embedded into the groove of the base die;

the outer walls of the outer layer pore plate die and the inner layer pore plate die are both provided with a plurality of embedding holes, and the horizontal channel die is inserted into the embedding holes; the molds are combined to simulate a multi-channel complex organ.

2. A combined mould for organ manufacture and drug screening according to claim 1, characterised in that: the base mould is rectangular, the bottom surface of the base mould is a plane, a concave surface or a convex surface, a cylindrical groove for fixing the vertical channel mould is arranged in the middle of the base mould, four rectangular edges of the base mould are respectively provided with a rectangular groove for fixing four outer-layer orifice plate moulds, and four grooves for fixing the inner-layer orifice plate mould are also arranged in the base mould;

the outer layer hole plate die comprises an outer layer hole plate die I, an outer layer hole plate die II, an outer layer hole plate die III and an outer layer hole plate die IV; the four outer layer pore plate moulds are embedded into the rectangular grooves of the corresponding base moulds;

the inner-layer pore plate die comprises a first inner-layer pore plate die, a second inner-layer pore plate die, a third inner-layer pore plate die and a fourth inner-layer pore plate die; the four inner-layer pore plate molds are embedded into the corresponding grooves of the base mold;

the horizontal channel molds are divided into two groups, each group comprises three layers, the two groups of channels are crossed into 90 degrees, and each layer is provided with six branches; the two groups of horizontal channels are fixed by inserting the corresponding embedding holes in the pore plate die, and the length of the horizontal channel die is greater than the distance between the two corresponding outer layer pore plate dies.

3. A combined mould for organ manufacture and drug screening according to claim 1, characterised in that: the complex organ is a complex organ structure with a middle large channel and a plurality of small channels and different cell matrix layers;

the five vertical channels are used for simulating thicker pipelines in organs, wherein the thicker pipelines comprise a trachea in a lung, a pancreatic duct in a pancreas and a bile duct in a liver;

the vertical channel mould adopts a spindle-shaped branch structure, and holes are punched on the pipe wall;

multiple horizontal channel molds are used to mimic the thinner conduits in an organ, including small vessels surrounding the middle arteriovenous vessels, nerve conduits, bronchial structures.

4. A combined mould for organ manufacture and drug screening according to claim 1, characterised in that: the two-layer structure is made of hydrogel materials and used for culturing specific tissues at different parts of an organ, and different pipelines can be used for culturing different cells.

5. A combined mould for organ manufacture and drug screening according to claim 4, characterised in that: the different cells include vascular epithelial cells, nerves, bronchial epithelial cells.

6. A combined mould for organ manufacture and drug screening according to claim 1, characterised in that: the base mold, the five vertical channel molds, the outer layer orifice plate mold, the horizontal channel mold and the inner layer orifice plate mold are all made of synthetic polymer materials or metal materials without biotoxicity.

7. A combined mould for organ manufacture and drug screening according to claim 1, characterised in that: the diameter of the horizontal channel die is 0.1 mm-5 mm; the diameter of the vertical channel die is the same as that of the corresponding groove on the base die; the groove of the base mold is the same as the thickness of the corresponding floor slab mold.

8. A method of using a combination mold for organ manufacture and drug screening, characterized by: the method comprises the following steps:

step 1, (extraction or purchase) preparation of a cell suspension from animal or human cells, the cell suspension having a density of 1X 10³～5×10⁷Preparing 1-30% of natural polymer hydrogel solution serving as a matrix solution, and mixing the matrix solution and the cell suspension according to a volume ratio of 1-9: 9-1, mixing to prepare a matrix solution containing cells, and obtaining a plurality of matrix solutions containing one type of cells by changing the selected cell type and the type of the matrix solution;

step 2, coating a layer of matrix solution containing cells on all channel molds, and obtaining a layer of tubular cell matrix layer by using a physical and chemical crosslinking or polymerization method, wherein the cell matrix layer on the surface of each tubular channel is ensured to have a certain thickness;

and 3, assembling all components of the combined die: firstly, fixing an inner layer of pore plate mould and an outer layer of pore plate mould, then inserting the horizontal channel mould which is subjected to cross-linking treatment in the step 2 into corresponding holes on the pore plate mould, and fixing five vertical channel moulds in grooves of a base mould; adding a matrix solution containing cells into the inner cavity of the mold consisting of the inner-layer pore plate to fully fill the whole inner cavity with the matrix solution; preparing an inner cell matrix layer by using a physical and chemical crosslinking or polymerization method; then the horizontal channel mould is withdrawn to the outer layer hole plate mould, the inner layer hole plate mould is taken out, and the horizontal channel mould is restored to the original position; then adding another matrix solution containing cells into the whole outer-layer inner cavity consisting of the outer-layer pore plate mould and the inner-layer pore plate mould to fully fill the whole inner cavity with the matrix solution;

preparing an outer cell matrix layer by using a physical and chemical crosslinking or polymerization method;

step 4, pulling out all the installed horizontal channel molds by using forceps, adding seed cells, culture medium and growth factors, and performing targeted culture to form an artificial organ containing vascular endothelial cells, and/or bronchial mucosa endothelial cells and/or nerve conduit endothelial cells;

and 5, dissolving the medicine in a cell culture solution or a PBS solution, injecting the medicine into a pipeline on the combined die, allowing the medicine to flow through the artificial organ containing the multicellular layer, regularly extracting the liquid in the pipeline, measuring the concentration change of the medicine, characterizing the cell shape, and identifying the reaction degree of the cell to the medicine.

9. The method of using a combination mold for organ manufacture and drug screening according to claim 8, wherein: the hydrogel solution is one or more of collagen, agarose, fibrinogen, sodium alginate, silk fibroin, gelatin, hyaluronic acid, chitosan, fibronectin, thrombin and polyethylene glycol.

10. The method of using a combination mold for organ manufacture and drug screening according to claim 8, wherein: the combined die can form a plurality of layers of cell matrix layers containing different cells to complete the assembly of various cell tissues;

adding cell growth factors and anticoagulant factors into the cell matrix solution, wherein the volume percentage of the cell growth factors and the anticoagulant factors in the total solution is 0.001-0.1%; the cell growth factor comprises one or more of endothelial cell growth factor, cell transfer factor or lung cell growth factor, and the anticoagulant factor comprises one or more of heparin, paclitaxel or sulfated chitosan.

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