A special mold and method for preparing living organs

Abstract

The present invention belongs to the field of artificial organ manufacturing technology, and in particular, relates to a special mold and a method for preparing living organs. It overcomes the difficulty of the prior art in realizing the precise construction of various shapes of artificial organs containing multi-branch vertical channels. It includes a bottom mold, at least one inner mold and at least one outer mold; the inner wall of the bottom mold forms an annular step with a radius increasing step by step from bottom to top, and a groove is arranged on the bottom surface of the bottom mold; the number of outer molds corresponds to the number of step layers, and the outer mold is a sleeve-shaped structure with two ends open; the outer mold is placed on the corresponding step to form an open-top structure; the number of inner molds is the same as the number of grooves, and each inner mold corresponds to a groove, and each inner mold includes a transverse mold for simulating secondary branch channels of blood vessels, bile ducts, nerves, and trachea, and a longitudinal mold for simulating primary channel branches; when in use, the longitudinal mold is embedded in the corresponding groove of the bottom mold, and the diameter of the groove is completely consistent with the diameter of the longitudinal mold.

Description

Technical Field

[0001] The present invention belongs to the technical field of artificial organ manufacturing, and in particular relates to a special mold and a method for preparing living organs; in particular, it relates to a special mold with more than one set of vertical branches and convenient assembly and disassembly in the manufacturing process of biological artificial organs with multi-branch vertical channels and a method for using the same. Background Art

[0002] At present, the occurrence of organ defects, failure or damage in the human body often leads to organ dysfunction and seriously affects the normal life of patients or even endangers their lives. At present, the common method for treating such diseases in clinical practice is organ transplantation, but the serious shortage of donor organs and the need to use immunosuppressive drugs for a long time to suppress the patient's immune rejection after organ transplantation have seriously restricted the application and development of organ transplantation technology. In order to solve the impact of end-stage organ damage diseases on people's health, the National Science Foundation of the United States formally established tissue engineering in 1987. As an emerging discipline that combines cell biology and materials science to construct tissues and organs in vitro or in vivo, tissue engineering can achieve in vitro culture of patients' own cells in its research field, and finally develop artificial organs that meet the patient's own needs. This can not only reduce the phenomenon of patients missing treatment opportunities due to insufficient donor organs, but also avoid immune rejection after organ transplantation, bringing new hope for the treatment of many patients with tissue defects and organ failure.

[0003] At present, the achievements of tissue engineering are mainly focused on the preparation of bone tissue and skin tissue with relatively simple structures, while the research on complex organs has been slow to make breakthroughs. The main reason is that complex organs often require more complex cell structures, as well as tubular network systems such as blood vessels, nerves, bile ducts, and trachea. In particular, the construction of vascular networks is a key link in the in vitro culture of complex tissues and organs. The vascular network plays an important role in providing sufficient oxygen and nutrients to the tissue cells of various parts of the organ while excreting metabolic waste from the body. Studies have shown that liver cells are easy to survive within a range of 100 to 200 μm around capillaries, otherwise they will soon die due to lack of oxygen and nutrients. The lack of a sufficiently fine vascular network will make it difficult to achieve the construction of larger tissues. In addition, some other tubular structures are also very important in organ construction. If these tubular structures cannot be well constructed, it will be difficult to maintain normal tissue structure and physiological function. Therefore, the construction of branched ducts such as blood vessels and nerves has become a research hotspot in the field of artificial organ manufacturing.

[0004] The invention patent (application number 202010361588.9) "Special combination mold for preparing complex organs and method for preparing liver precursors" proposes a combination mold that mimics the structure of liver lobule, which is used to prepare liver precursors with blood vessels and bile duct networks and introduces the preparation method. However, this special combination mold has only one primary channel and cannot prepare different types of pipes at the same time. In addition, the length of the small branches is fixed and cannot be adjusted according to the size of the external mold. It is only suitable for preparing liver precursors and is not ideal for the preparation of other complex organs.

[0005] The invention patent (application number 201910682212.5) "Detachable special mold and method for preparing artificial organ precursors with multiple branch channels" proposes a special mold for preparing artificial organ precursors with multiple branch channels and a corresponding preparation method. The internal channel mold of the special mold is detachable, and it will not damage the formed structure when it is taken out from the formed cell matrix layer, but there are problems such as difficult disassembly operation and too small feeding port, which makes operation inconvenient.

[0006] The invention patent (application number 201510419730.X) "A method for preparing a tissue organ precursor with multiple branch channels and a special mold" proposes a method for preparing a tissue organ precursor with multiple branch channels and a special mold. The disadvantage is that there are too many intermediate steps and the large and small channels are easily misaligned. If the added hydrogel solution has a certain viscosity, it is difficult to fill the entire inner cavity. When the one-piece branch inner mold is taken out from the cell matrix layer, it will damage the formed cell matrix layer. Summary of the invention

[0007] The present invention is aimed at the defects of the prior art and provides a special mold and a method for preparing a living organ. The purpose is to use an inner mold and a multi-level outer mold that are easy to combine and disassemble to form an artificial organ with multi-branched vertical channels with different cell matrix layers on the basis of existing work, so as to overcome the difficulty of the prior art in realizing the precise construction of various shapes of artificial organs with multi-branched vertical channels.

[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical scheme, including a bottom mold, at least one inner mold and at least one outer mold; the inner wall of the bottom mold forms a (multi-layer) annular step with a radius increasing step by step from bottom to top, and a groove is arranged on the bottom surface of the bottom mold; the number of outer molds corresponds to the number of step layers, and the outer mold is a sleeve-shaped structure (or annular structure) with open ends; the outer mold is placed on the corresponding step to form an open-top structure; the number of inner molds is the same as the number of grooves, each inner mold corresponds to a groove, and each inner mold includes a transverse mold for simulating secondary branch channels of blood vessels, bile ducts, nerves, and trachea, and a longitudinal mold for simulating primary channel branches; when in use, the longitudinal mold is embedded (inserted) into the corresponding groove of the bottom mold, and the diameter of the groove is completely consistent with the diameter of the longitudinal mold.

[0009] Furthermore, there are three steps and three outer molds, and all of the outer molds are sleeve-shaped structures (or ring-shaped structures) open at both ends; the three outer molds are: an inner outer mold, a middle outer mold, and an outer outer mold; the three outer molds are placed on corresponding steps; the lower-level outer mold is seamlessly fitted with its adjacent upper-level outer mold; the contour of the inner cavity of each layer of the outer mold is consistent with the shape of the artificial organ to be manufactured.

[0010] Furthermore, the transverse mold is inserted into (or tightly plugged into) a hole reserved in the longitudinal mold.

[0011] Furthermore, the inner outer mold is placed on the first step, the middle outer mold is placed on the second step, and the outer outer mold is placed on the third step; and after placement, the centers of the three outer molds are collinear and the tops of the outer molds are flush.

[0012] Furthermore, the outer mold and its corresponding step fit seamlessly at the contact point.

[0013] Furthermore, the bottom surface of the bottom mold can be any one of a flat surface, a concave surface or a convex surface, and the two grooves are two circular grooves with the same size.

[0014] Furthermore, the cross-section of the longitudinal mold can be any one of circular, square, and elliptical shapes, and the holes on the longitudinal mold are consistent with the cylindrical diameter of the transverse mold; the outer mold is made of hard non-biotoxic material (including synthetic polymers), and the inner mold is made of non-biotoxic material (including any one of synthetic fibers, polyethylene, polyvinyl chloride or photosensitive resin).

[0015] Furthermore, after the transverse mold passes through the hole on the longitudinal mold, the specific position can be adjusted in the horizontal direction (with a different penetration depth relative to the longitudinal mold) to cooperate with the outer molds with different inner diameters.

[0016] The special mold and the method for preparing living organs are characterized by comprising the following steps:

[0017] 1) Prepare a hydrogel solution with a mass volume concentration of 0.5% to 20% as the matrix solution, extract or purchase animal cells to make a cell suspension, and the cell density is 1×10 3 ~5×10 7 The matrix solution is mixed with the cell suspension at a volume ratio of 1:9 to 9:1 to prepare a matrix solution containing cells. The above steps are repeated to prepare a matrix solution containing multiple (eg, three) different cells.

[0018] 2) Prepare a synthetic polymer solution with a mass percentage concentration of 1% to 30%, wherein the synthetic polymer material is a composite of one or more of polyurethane, polycaprolactone, polycarbonate, polylactic acid, polyethylene glycol, and polyhydroxy acid ester, and the organic solvent used to dissolve the synthetic polymer material is tetraethylene glycol, ethylene glycol, isopropanol or 1,4-dioxane.

[0019] 3) Use 3D printing technology to print non-cytotoxic materials into various components of a special mold; pass four horizontal molds through the holes on the vertical mold to form a set of inner molds; according to different needs, a layer of cell-containing matrix solution can be first coated on the inner mold to form structures such as blood vessels, bile ducts, nerves, and trachea; then install the longitudinal mold in the group of inner molds into a positioning groove on the bottom mold to complete the positioning of the group of inner molds; then assemble another group of inner molds in the same way and coat them according to needs to form a structure different from the first group of inner molds, and install them in another positioning groove on the bottom mold; install the inner outer mold on the inner step of the bottom mold, and adjust the position of the horizontal mold in the horizontal direction so that one end of it is close to the inner wall of the inner outer mold. A cell-containing matrix solution is added to the inner cavity of the mold from the top, and the first cell matrix layer is formed by physical or chemical cross-linking.

[0020] 4) Remove the inner outer mold, first take out the lateral inner mold in the horizontal direction, then install the middle outer mold on the corresponding step of the bottom mold, add another cell matrix solution into the cavity between the middle inner mold and the formed cell matrix layer through the upper end of the outer mold, and cross-link to form the second cell matrix layer.

[0021] 5) Remove the middle outer mold, install the outer outer mold on the corresponding step of the bottom mold, and repeat the adding steps in step 4) to prepare the third cell matrix layer.

[0022] 6) Remove the outer outer mold and the bottom mold, and dismantle the longitudinal mold along the vertical direction. This can reduce the damage to the formed cell matrix layer, remove the formed cell matrix layer, and finally obtain a relatively complete complex living organ with multi-branched vertical channels.

[0023] Furthermore, the hydrogel solution is made of one or more of the materials such as gelatin, sodium alginate, collagen, agarose, hyaluronic acid, fibrinogen, chitosan, silk fibroin, pluronic, polyethylene glycol, etc., and the hydrogel solution obtained by mixing multiple materials has a better effect.

[0024] Furthermore, multiple cell matrix layers containing different cells can be formed to complete the assembly of various cell tissues.

[0025] Furthermore, before or after the formation of the artificial organ precursor, one or more of endothelial cell growth factor, cell transfer factor or hepatocyte growth factor, as well as one or more of heparin, paclitaxel or sulfated chitosan can be added to the multi-branched vertical channel structure to induce the differentiation of channel surface cells to form tubular networks such as blood vessels, bile ducts, trachea, nerves, etc.

[0026] Furthermore, before perfusing the cells, it is ensured that the two groups of inner molds are not connected to each other and do not interfere with each other, and during coating, it is ensured that the cell matrix layer on the surface of each tubular channel has a certain thickness.

[0027] Compared with the prior art, the present invention has beneficial effects.

[0028] The present invention adopts a combination mold that is easy to assemble and disassemble to prepare a living organ with multi-branch vertical internal channels. The secondary channels formed by the horizontal molds in the two sets of inner molds are not connected to each other, and two vertical channels of the same or different types can be formed. Different cell matrix layers are obtained by layered perfusion. The molds can be freely combined and are easy to assemble and disassemble. The damage to the formed cell matrix layer is minimized during demoulding. The size and shape of the inner and outer bottom molds can be adjusted according to the organ precursor to be formed. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. The protection scope of the present invention is not limited to the following description.

[0030] Figure 1 It is an overall top view of the special mold.

[0031] Figure 2 This is the appearance of the bottom mold.

[0032] Figure 3 It is a schematic diagram of the structure of a set of inner molds.

[0033] Figure 4 This is a diagram of the formation of an artificial organ with multi-branched vertical channels (taking an organ containing three different cell matrix layers as an example).

[0034] Figure 5 It is an overall schematic diagram of the special mold (the dotted line is the blocked part).

[0035] In the figure, 101 is a longitudinal mold, 102 is a transverse mold, 201 is an inner outer mold, 202 is a middle outer mold, 203 is an outer outer mold, 301 is a bottom mold, 401 is a first cell matrix layer, 402 is a second cell matrix layer, 403 is a third cell matrix layer, and 404 is a hollow tube. DETAILED DESCRIPTION

[0036] like Figure 1-5 As shown, 401 is a first cell matrix layer; 402 is a second cell matrix layer; 403 is a third cell matrix layer; 404 is a hollow tube used for constructing tubular or branch-like networks such as blood vessels, bile ducts, trachea, urethra, and nerves.

[0037] The special mold described in the present invention includes a bottom mold, at least one group of inner molds and at least one group of outer molds. Among them, the inner mold is realized by embedding the horizontal mold simulating the secondary branch channels of blood vessels, bile ducts, nerves, and trachea into the corresponding holes reserved on the longitudinal mold simulating the primary channel branches. The inner mold is fixed by embedding the longitudinal mold into the corresponding groove on the bottom mold. The longitudinal mold is in the shape of a cylinder, a square, or an ellipse, and has a group (such as four) of circular holes on it. The diameters of these holes are consistent with the cylindrical diameters of the horizontal molds, so that the gap between the two after the horizontal molds are embedded in the longitudinal mold is extremely small. Each group of horizontal molds is embedded with a longitudinal mold to form a group of inner molds. The bottom mold is stepped from bottom to top, and the number of three steps corresponds to the number of three outer molds. The bottom surface of the bottom mold can be a plane, a concave surface, or a convex surface. Two small circular grooves of exactly the same size are distributed on the bottom mold, and the diameter of the grooves is exactly the same as the diameter of the longitudinal mold. The axes of the three-level outer molds are located in the same straight line, and the outer dimensions of the lower end of each level of the outer mold are the same as the surface dimensions enclosed by the outer surfaces of the corresponding level of steps of the bottom mold. The inner cavity of the next level of the outer mold is consistent in size with the outer cavity of the previous level of the outer mold, and the contour of the inner cavity of each level of the outer mold can be designed to be consistent with the shape of the artificial organ to be manufactured. The upper part of each group of outer molds is open for easy feeding.

[0038] The special mold and the method for preparing living organs are as follows: the outer mold is made of hard non-biotoxic materials, such as synthetic polymers, and the inner mold is made of non-biotoxic materials to form a corresponding solid tubular mold. These materials can be synthetic fibers, polyethylene, polyvinyl chloride or photosensitive resins, but are not limited to these materials.

[0039] The special mold and the method for preparing living organs thereof, the inner mold is composed of two-stage detachable molds, when disassembling, the horizontal mold can be removed first and then the vertical mold, reducing the damage to the formed cell matrix layer.

[0040] A special mold and a method for preparing a living organ thereof include the following steps:

[0041] 1) Prepare a hydrogel solution with a mass volume concentration of 0.5% to 200% as the matrix solution, extract or purchase animal cells to make a cell suspension, and the cell density is 1×10 3 ~5×10 7 / ml, the matrix solution and the cell suspension are mixed at a volume ratio of 1:9 to 9:1 to prepare a matrix solution containing cells, and the above steps are repeated to prepare three matrix solutions containing different cells. The hydrogel solution can generally be made of one or more of gelatin, sodium alginate, collagen, agarose, hyaluronic acid, fibrinogen, chitosan, silk fibroin, pluronic, polyethylene glycol and other materials. The hydrogel solution obtained by mixing multiple materials is better.

[0042] 2) Prepare a synthetic polymer solution with a mass percentage concentration of 1% to 30%, wherein the synthetic polymer material is a composite of one or more of polyurethane, polycaprolactone, polycarbonate, polylactic acid, polyethylene glycol, and polyhydroxy acid ester, and the organic solvent used to dissolve the synthetic polymer material is tetraethylene glycol, ethylene glycol, isopropanol or 1,4-dioxane.

[0043] 3) Use 3D printing technology to print non-cytotoxic materials into various components of a special mold. Four transverse molds are installed in the holes of the longitudinal mold to form a set of inner molds. According to different needs, a layer of matrix solution containing cells can be first coated on the inner mold to form structures such as blood vessels, bile ducts, nerves, and trachea. Then the longitudinal mold in the group of inner molds is installed in a positioning groove on the bottom mold to complete the positioning of the group of inner molds. Then assemble another group of inner molds in the same way, and coat them according to needs to form a structure different from the first group of inner molds, and install them in another positioning groove on the bottom mold. Install the inner outer mold on the inner step of the bottom mold, and adjust the position of the transverse mold in the horizontal direction so that one end of it is close to the inner wall of the inner outer mold. A matrix solution containing cells is added to the inner cavity of the mold from the top, and the first layer of cell matrix layer is formed by physical or chemical cross-linking. Before perfusing cells, ensure that the two groups of inner molds are not connected to each other and do not interfere with each other.

[0044] 4) Remove the inner outer mold and the lateral inner mold, install the larger middle outer mold on the corresponding step of the bottom mold, add the matrix solution containing the second cell into the cavity between the middle inner mold and the formed cell matrix layer through the feeding port, and cross-link to form the second cell matrix layer.

[0045] 5) Remove the middle outer mold, install the outer outer mold on the corresponding step of the bottom mold, and repeat the adding steps in step 3) to prepare the third cell matrix layer.

[0046] 6) Remove the outer mold and the bottom mold, and dismantle the longitudinal mold. This can reduce the damage to the formed cell matrix layer and ultimately obtain a relatively complete living organ with multiple branched channels.

[0047] 7) Before or after the formation of the organ precursor, one or more of endothelial cell growth factor, cell transfer factor or hepatocyte growth factor, as well as one or more of heparin, paclitaxel or sulfated chitosan can be added to the multi-branched channel structure to induce the differentiation of channel surface cells into tubular or dendritic networks such as blood vessels, bile ducts, trachea, and nerves.

[0048] Example 1: Preparation of a living liver with multiple branched blood vessels and bile ducts.

[0049] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are used to print various components of the special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and one outer mold of different levels. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0050] 2) 1% gelatin and 2% sodium alginate were mixed and dissolved in PBS solution as matrix solution 1, 2% sodium alginate and 1% fibrinogen were mixed and dissolved in PBS solution as matrix solution 2, 20 IU / mL thrombin solution and 2% calcium chloride solution were prepared, and adipose stem cells, endothelial cell growth factor, bile duct epithelial cells, hepatic stellate cells, sinusoidal endothelial cells and hepatocytes were purchased or extracted to prepare cell suspensions, with a cell density of 1×10 6 / ml, prepared with a 10% (w / v) polyurethane / tetraethylene glycol solution as the synthetic polymer solution, and added with 1% (w / v) heparin.

[0051] 3) A group of inner molds are subjected to vascularization treatment to form a vascular inner mold, specifically, the matrix solution 2 is mixed with a cell suspension containing adipose stem cells and endothelial cell growth factor in a volume ratio of 1:1 to form a cell-containing matrix solution, and the solution is coated on the surface of the inner mold, and the calcium chloride solution and thrombin solution are used to chemically cross-link to form a vascular structure. The matrix solution 2 is mixed with a cell suspension containing bile duct epithelial cells in a volume ratio of 1:2 to form a cell-containing matrix solution, and the solution is coated on the surface of another group of inner molds, and the calcium chloride solution and thrombin solution are used to chemically cross-link to form a bile duct structure. The above coating should be sufficient so that the cell-containing matrix layer on each group of inner molds has a certain thickness.

[0052] 4) Insert the longitudinal mold in each group of inner molds into the positioning groove of the bottom mold, keep a certain distance between the two groups of inner molds, and then insert the inner outer mold into the first step of the bottom mold. The cell matrix solution prepared by mixing the matrix solution 2 and the cell suspension containing hepatocytes in a volume ratio of 1:1 is poured into the inner cavity of the mold through the opening at the upper end of the outer mold, and calcium chloride and thrombin solution are added successively to cross-link them to form the first stable cell matrix layer, which contains a group of vascular channels and a group of bile duct channels.

[0053] 5) Remove the inner outer mold and the lateral mold, install the middle outer mold on the second step of the bottom mold, mix the matrix solution 1 with the cell suspension containing hepatocytes and hepatic stellate cells in a volume ratio of 1:1 to prepare a cell matrix solution, inject it into the inner cavity of the mold through the opening at the upper end of the outer mold, and then perform chemical cross-linking with a calcium chloride solution to form another stable cell matrix layer.

[0054] 6) The middle outer mold is removed, and the outer outer mold is installed on the third step of the bottom mold. The cell matrix solution prepared by mixing the matrix solution 1 with the cell suspension containing hepatocytes and hepatic sinusoidal endothelial cells in a volume ratio of 1:1 is poured into the inner cavity of the mold through the feeding port, and then chemically cross-linked with a calcium chloride solution to form a third stable cell matrix layer.

[0055] 7) Spraying the synthetic polymer solution onto the surface of the formed multicellular matrix layer to form a synthetic polymer material layer after extraction, and removing the base mold.

[0056] 8) The longitudinal inner mold is slowly pulled out to obtain a relatively complete living liver with multiple different cell matrix layers while minimizing damage to the cell matrix layer.

[0057] Example 2: Preparation of a living heart with multiple branched blood vessels and nerves.

[0058] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are printed into various components of a special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and an inner outer mold. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0059] 2) 2% fibrinogen and 1% agarose were mixed and dissolved in PBS as matrix solution 1, 5% gelatin solution was prepared as matrix solution 2, 20 IU / mL thrombin solution was prepared, endothelial cells, hemangioblastoma cells and cardiomyocytes were purchased to prepare cell suspensions. The cell density of endothelial cell suspension and hemangioblastoma cell suspension was 1×10 6 The cell density of myocardial cell suspension was 1×10 5The matrix solution and the cell suspension were mixed in a volume ratio of 1:1 to prepare a cell-containing matrix solution, a 15% (w / v) polyurethane / tetraethylene glycol solution was prepared as a synthetic polymer solution, and 2% (w / v) paclitaxel was added.

[0060] 3) A group of inner molds are subjected to vascularization treatment to form vascular inner molds. Specifically, a cell-containing matrix solution is prepared by mixing matrix solution 1 and a cell suspension containing endothelial cells in a volume ratio of 1:2, and coated on the surface of a group of inner molds, and chemically cross-linked with a thrombin solution to form a vascular structure. The coating should be sufficient so that the cell-containing matrix layer on the inner mold has a certain thickness. Another group of inner molds are not treated and used as neural inner molds.

[0061] 4) Insert the longitudinal mold in each group of inner molds into the positioning groove of the bottom mold, keep a certain distance between the two groups of inner molds, and then insert the inner outer mold into the first step of the bottom mold. The cell matrix solution prepared by mixing the matrix solution 1 and the cell suspension containing myocardial cells in a volume ratio of 1:1 is poured into the inner cavity of the mold through the opening at the upper end of the outer mold, and the thrombin solution is added to chemically cross-link it to form a stable cell matrix layer, which contains a group of vascular channels and a group of neural tubular channels.

[0062] 5) Remove the inner outer mold and the lateral mold, spray the synthetic polymer solution onto the surface of the formed cell matrix layer, form a synthetic polymer material layer after extraction, and remove the base mold.

[0063] 6) Slowly pull out the longitudinal mold to minimize damage to the cell matrix layer.

[0064] 7) The matrix solution 2 and the cell suspension containing hematopoietic cells are mixed in a volume ratio of 1:1 to prepare a cell-containing matrix solution, which is perfused into the previously reserved nerve channel. After 20 minutes of physical cross-linking at a temperature below 37°C, the solution is formed into a neural tubular pathway, thereby obtaining a relatively complete living heart with multiple branched blood vessels and nerves.

[0065] Example 3: Preparation of a living bladder with multi-branched blood vessels and urethra.

[0066] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are printed into various components of a special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and an inner outer mold. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0067] 2) Prepare a mixed solution containing 2% gelatin and 3% sodium alginate as the matrix solution, 2% calcium chloride solution, and purchase endothelial cells, urethral epithelial cells, and bladder cells to make a cell suspension. The cell density is 1×10 6 The matrix solution and the cell suspension were mixed in a volume ratio of 1:1 to prepare a cell-containing matrix solution, prepared with 20% polyurethane / tetraethylene glycol solution as a synthetic polymer solution, and added with 1% paclitaxel.

[0068] 3) A group of inner molds are treated with vascularization to form a vascular inner mold. The specific method is to apply a layer of matrix solution containing endothelial cells on the surface of one group of inner molds, and chemically cross-link them with a calcium chloride solution to form a vascular structure. Another group of inner molds are treated to be used as urethral inner molds. The specific method is to apply a layer of matrix solution containing urethral epithelial cells on the surface of the inner molds, and chemically cross-link them with a calcium chloride solution to form a urethral structure. The above coating should be sufficient so that the cell-containing matrix layer on each group of inner molds has a certain thickness.

[0069] 4) Insert the longitudinal mold in each set of inner molds into the positioning groove of the bottom mold, keep a certain distance between the two sets of inner molds, and then insert the inner outer mold into the first step of the bottom mold. Inject the cell matrix solution containing bladder cells into the inner cavity of the mold through the upper opening of the outer mold, add calcium chloride solution for chemical cross-linking, and form the first stable cell matrix layer, which contains a group of vascular channels and a group of urethral channels.

[0070] 5) Remove the inner outer mold and the lateral mold, spray the synthetic polymer solution onto the surface of the formed cell matrix layer, form a synthetic polymer material layer after extraction, and remove the base mold.

[0071] 6) Slowly pull out the longitudinal mold to damage the cell matrix layer as little as possible, thereby producing a relatively complete living bladder with multiple branched blood vessels and urethra.

[0072] Example 4: Preparation of living lungs with multi-branched blood vessels and trachea.

[0073] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are printed into various components of a special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and a middle outer mold. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0074] 2) Use 5% fibrinogen solution as the matrix solution; prepare 20 IU / mL thrombin solution, purchase endothelial cells and lung cells to make cell suspension, and the cell density is 5×10 cells / ml; mix the matrix solution and the cell suspension in a volume ratio of 1:2 to make a cell-containing matrix solution; prepare 5% polyurethane / ethylene glycol solution as the synthetic polymer solution, and add 1% paclitaxel.

[0075] 3) A group of inner molds are vascularized to form vascular inner molds. The specific method is to apply a layer of matrix solution containing endothelial cells on the surface of one group of inner molds, and use thrombin solution for chemical cross-linking to form a vascular structure. The coating should be sufficient so that the cell-containing matrix layer on the inner mold has a certain thickness. The other group of inner molds is used as trachea and is not processed for the time being.

[0076] 4) Insert the longitudinal mold in each set of inner molds into the positioning groove of the bottom mold, keep a certain distance between the two sets of inner molds, and then insert the middle outer mold into the second step of the bottom mold. Infuse the cell matrix solution containing lung cells into the inner cavity of the mold through the upper opening of the outer mold, add thrombin solution for chemical cross-linking, and form a stable cell matrix layer, which contains a group of vascular channels and a group of tracheal channels.

[0077] 5) The middle outer mold is removed, and the lateral mold is dismantled, and the synthetic polymer solution is sprayed onto the surface of the formed cell matrix layer to form a synthetic polymer material layer after extraction, and the base mold is removed.

[0078] 6) The longitudinal inner mold is slowly pulled out to damage the cell matrix layer as little as possible, thereby producing a relatively complete living lung with multiple branched blood vessels and trachea.

[0079] Example 5: Preparation of a living breast with multi-branched blood vessels and milk ducts.

[0080] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are printed into various components of a special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and a middle outer mold. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0081] 2) Dissolve 5% gelatin and 1% agarose in PBS solution as matrix solution, extract or purchase adipose stem cells and mammary epithelial cells, purchase endothelial cell growth factor and adipocyte growth factor, and mix the two growth factors with adipose stem cells to prepare cell suspension. The cell density is 1×10 7 The mammary epithelial cells were prepared into cell suspension with a cell density of 1×10 6The matrix solution and the cell suspension were mixed in a volume ratio of 1:1 to prepare a cell-containing matrix solution, a 10% (w / v) polyurethane / tetraethylene glycol solution was prepared as a synthetic polymer solution, and 1% (w / v) heparin was added.

[0082] 3) A group of inner molds are subjected to vascularization treatment to form vascular inner molds, specifically by coating a layer of matrix solution containing adipose stem cells and endothelial cell growth factor on the surface of the inner mold, and then physically crosslinking at a temperature below 37°C for 20 minutes to form an endothelial cell layer. Another group of inner molds are subjected to ductalization treatment to form ductal inner molds, specifically by coating a layer of matrix solution containing mammary epithelial cells on the surface of the inner mold, and then physically crosslinking at a temperature below 37°C for 20 minutes to form a ductal structure. The coating should be sufficient so that the cell-containing matrix layer on the inner mold has a certain thickness.

[0083] 4) Insert the longitudinal inner mold in each group of inner molds into the positioning groove of the bottom mold, keep a certain distance between the two groups of inner molds, and then insert the middle outer mold into the second step of the bottom mold. Inject the cell matrix solution containing adipose stem cells and adipose cell growth factor into the inner cavity of the mold through the upper opening of the outer mold, and after 20 minutes of physical cross-linking at below 37°C, a stable cell matrix layer is formed, which contains a group of vascular channels and a group of milk duct channels.

[0084] 5) Remove the middle outer mold and the lateral inner mold, spray the synthetic polymer solution onto the surface of the formed cell matrix layer, form a synthetic polymer material layer after extraction, and remove the base mold.

[0085] 7) The longitudinal mold is slowly pulled out to obtain a relatively complete living breast with multiple branched blood vessels and trachea while minimizing damage to the cell matrix layer.

[0086] Example 6: Preparation of a living brain with multi-branched blood vessels and nerves.

[0087] 1) Using 3D printing technology, non-cytotoxic photosensitive resin materials are printed into various components of a special mold, including 2 longitudinal molds, 8 transverse molds, 1 three-level bottom mold, and an outer mold. The four transverse molds are installed into the holes on the longitudinal mold to form a set of inner molds.

[0088] 2) Dissolve 3% gelatin and 1% fibrinogen in PBS solution as matrix solution, prepare 20 IU / mL thrombin solution, extract or purchase adipose stem cells and brain glial cells, purchase endothelial cell growth factor and neural cell growth factor, and mix the two growth factors with adipose stem cells to make cell suspension, with a cell density of 1×10 5Brain glial cells were prepared into cell suspension with a cell density of 1×10 6 The matrix solution and the cell suspension were mixed in a volume ratio of 1:1 to prepare a cell-containing matrix solution, a 10% (w / v) polylactic acid / tetraethylene glycol solution was prepared as a synthetic polymer solution, and 1% (w / v) heparin was added.

[0089] 3) A group of inner molds are subjected to vascularization treatment to form vascular inner molds, specifically by coating a layer of matrix solution containing adipose stem cells and endothelial cell growth factor on the surface of the inner mold, and chemically cross-linking with thrombin solution to form an endothelial cell layer. Another group of inner molds are subjected to neuralization treatment, specifically by coating a layer of matrix solution containing adipose stem cells and nerve cell growth factor on the surface of another group of molds, and chemically cross-linking with thrombin solution to form a nerve cell layer. The coating should be sufficient so that the cell-containing matrix layer on the inner mold has a certain thickness.

[0090] 4) Insert the longitudinal inner mold in the inner mold assembly prepared above into the positioning groove of the bottom mold, keep a certain distance between the two sets of inner molds, and then insert the outer outer mold onto the third step of the bottom mold. Infuse the cell matrix solution containing brain glial cells into the inner cavity of the mold through the upper opening of the outer mold, add thrombin to make it cross-linked, and form a stable brain glial cell matrix layer, which contains a group of vascular channels and a group of neural channels.

[0091] 5) Spraying the synthetic polymer solution onto the surface of the formed cell matrix layer, forming a synthetic polymer material layer after extraction, and removing the bottom mold.

[0092] 6) The longitudinal mold is slowly pulled out to obtain a relatively complete living brain with multiple different cell matrix layers while minimizing damage to the cell matrix layer.

[0093] It can be understood that the above specific description of the present invention is only used to illustrate the present invention and is not limited to the technical solutions described in the embodiments of the present invention. Those skilled in the art should understand that the present invention can still be modified or replaced by equivalents to achieve the same technical effects; as long as the use requirements are met, they are within the protection scope of the present invention.

Claims

1. A method for preparing living organs using a special mold, It is characterized in that The special mold includes a bottom mold, at least one inner mold and at least one outer mold; The inner wall of the bottom mold forms an annular step with a radius increasing from bottom to top, and a groove is arranged on the bottom surface of the bottom mold; the number of the outer molds corresponds to the number of the step layers, and the outer mold is a sleeve-shaped structure with both ends open; the outer mold is placed on the corresponding step to form an open-top structure; the number of the inner molds is the same as the number of the grooves, and each inner mold corresponds to a groove, and each inner mold includes a transverse mold for simulating secondary branch channels of blood vessels, bile ducts, nerves, and trachea, and a longitudinal mold for simulating primary channel branches; when in use, the longitudinal mold is embedded in the corresponding groove of the bottom mold, and the diameter of the groove is completely consistent with the diameter of the longitudinal mold; A method for preparing a living organ, comprising the steps of: Step 1) Prepare a hydrogel solution with a mass volume concentration of 0.5% to 20% as a matrix solution, extract or purchase animal cells to make a cell suspension, and the cell density is 1×10 3 ~5×10 7 / ml, the matrix solution and the cell suspension are mixed at a volume ratio of 1:9 to 9:1 to prepare a matrix solution containing cells, and the above steps are repeated to prepare matrix solutions containing a variety of different cells; Step 2) preparing a synthetic polymer solution with a mass percentage concentration of 1% to 30%, wherein the synthetic polymer material is a composite of one or more materials selected from polyurethane, polycaprolactone, polycarbonate, polylactic acid, polyethylene glycol, and polyhydroxy acid ester, and the organic solvent used to dissolve the synthetic polymer material is tetraethylene glycol, ethylene glycol, isopropanol, or 1,4-dioxane; Step 3) Use 3D printing technology to print non-cytotoxic materials into various components of a special mold; pass four horizontal molds through the holes on the vertical mold to form a group of inner molds; according to different needs, a layer of cell-containing matrix solution can be first coated on the inner mold to form structures such as blood vessels, bile ducts, nerves, and trachea; then install the longitudinal mold in the group of inner molds into a positioning groove on the bottom mold to complete the positioning of the group of inner molds; then assemble another group of inner molds in the same way, and coat them according to needs to form a structure different from the first group of inner molds, and install them in another positioning groove on the bottom mold; install the inner outer mold on the inner step of the bottom mold, and adjust the position of the horizontal mold in the horizontal direction so that one end of it is close to the inner wall of the inner outer mold; add a cell-containing matrix solution into the inner cavity of the mold from the top, and use physical or chemical cross-linking to form a first cell matrix layer; Step 4) removing the inner outer mold, first taking out the lateral inner mold along the horizontal direction, and then installing the middle outer mold on the corresponding step of the bottom mold, adding another cell matrix solution into the cavity between the middle inner mold and the formed cell matrix layer through the upper end of the outer mold, and cross-linking to form a second cell matrix layer; Step 5) remove the middle outer mold, install the outer outer mold on the corresponding step of the bottom mold, and repeat the material adding step of step 4) to prepare the third cell matrix layer; Step 6) removing the outer outer mold and the bottom mold, and dismantling the longitudinal mold along the vertical direction, so as to reduce the damage to the formed cell matrix layer, remove the formed cell matrix layer, and finally obtain a relatively complete complex living organ with multi-branched vertical channels; The horizontal mold is inserted into the hole reserved on the vertical mold; The inner outer mold is placed on the first step, the middle outer mold is placed on the second step, and the outer outer mold is placed on the third step; and after placement, the centers of the three outer molds are collinear and the tops of the outer molds are flush; The outer mold and its corresponding step fit seamlessly at the contact point; The bottom surface of the bottom mold can be any one of a flat surface, a concave surface or a convex surface, and the two grooves are two circular grooves of the same size; The cross section of the longitudinal mold can be any of circular, square, and elliptical shapes, and the hole on the longitudinal mold is consistent with the cylinder diameter of the transverse mold; The outer mold is made of hard non-biotoxic material, and the inner mold is made of non-biotoxic material; After the transverse die passes through the hole on the longitudinal die, the specific position can be adjusted in the horizontal direction to match the outer die with different inner diameters; The hydrogel solution is made of one or more of the materials such as gelatin, sodium alginate, collagen, agarose, hyaluronic acid, fibrinogen, chitosan, silk fibroin, pluronic, polyethylene glycol, etc.; Able to form multiple cell matrix layers containing different cells and complete the assembly of various cell tissues; Before or after the artificial organ precursor is formed, one or more of endothelial cell growth factor, cell transfer factor or hepatocyte growth factor, as well as one or more of heparin, paclitaxel or sulfated chitosan can be added to the multi-branched vertical channel structure to induce the differentiation of channel surface cells to form tubular networks such as blood vessels, bile ducts, trachea, nerves, etc. Before perfusing cells, ensure that the two groups of inner molds are not connected to each other and do not interfere with each other, and during coating, ensure that the cell matrix layer on the surface of each tubular channel has a certain thickness.

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