WO2016041238A1 - 利用旋转堆积法制备组织器官的方法及专用设备 - Google Patents

利用旋转堆积法制备组织器官的方法及专用设备 Download PDF

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WO2016041238A1
WO2016041238A1 PCT/CN2014/089769 CN2014089769W WO2016041238A1 WO 2016041238 A1 WO2016041238 A1 WO 2016041238A1 CN 2014089769 W CN2014089769 W CN 2014089769W WO 2016041238 A1 WO2016041238 A1 WO 2016041238A1
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cells
cell
hydrogel
solution
nozzle
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王小红
周新伟
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body

Definitions

  • tissue engineering structures such as electrospinning, die casting, etc.
  • Cell viability is low, making it difficult to construct large pieces of tissue.
  • cells or cell aggregates
  • sols precursors of hydrogels
  • the deposition position of the cell-containing droplets is controlled by the computer.
  • Print one by one at a specified position continue printing another layer on the basis of printing one layer, and superimpose layers to form a three-dimensional multi-cell gel system.
  • Cell printing technology is characterized by uniformity of cell density and distribution, as well as the ability to co-assemble and position a variety of cells.
  • the problem of efficient delivery of oxygen and nutrients, the "vascularization problem” remains a bottleneck in the construction of transplantable complex tissues and organs.
  • the existing tissue and organs formed by 3D printing of cells are stacked according to the plane of one layer. Due to the inherent characteristics of the formation, the formation of microchannels (blood vessels) depends on the forming precision and the properties of the materials. And for having a lumen structure The formation of tissue and organ, relying on the planar deposition of cell printing technology inevitably requires the addition of brackets or rotating forming table or nozzle orientation during the forming process, greatly increasing the difficulty of design and forming, and sometimes even powerless.
  • Chinese patent document CN1654028 proposes a method for manufacturing a tubular mesh stent by using a lysing method, which firstly uses a water-soluble, non-biotoxic material to manufacture the inner core of the stent, and then applies a biocompatible material to the inner core of the stent. After air drying, the inner core of the stent was dissolved in distilled water. The method can only form a tubular mesh stent with a simple structure, and is incapable of forming a tissue organ with complex blood vessels.
  • the object of the present invention is to overcome the difficulty in constructing a cavity-like organ of a complex blood vessel and a nerve structure in a conventional tissue engineering, and to provide a method and a special device for preparing a tissue organ based on a rotational stacking method.
  • the method is based on the cell-controlled assembly technology and the core-dissolving technology, and aims to provide a new preparation method of tissue and organs, which is convenient for preparing tissues and organs having spiral reticulated blood vessels and nerves, and is particularly suitable for preparing a cavity structure. Organs and organs.
  • a method for preparing a tissue organ by a spin stacking method characterized in that the special device of the present invention comprises a supporting device, a multi-head assembly, a moving mechanism, a forming device and a control system; the supporting device comprises a supporting leg and a bottom plate;
  • a guide rail-slider mechanism is adopted, including an X-direction guide rail, an X-direction slide rail, a Y-direction guide rail, a first Y-direction slide, a second Y-direction slide, a Z-direction guide rail, and a Z-direction slide rail, wherein the X-direction guide rail is provided On the bottom plate, the Z-direction rail is fixed on the X-direction slider, and the Y-direction rail is fixed on the Z-direction slider;
  • the multi-head assembly comprises at least six nozzles, including at least two squeeze nozzles and four The spray nozzle has two nozzles having inner diameters of 200 ⁇ m to 1 mm and 50 ⁇
  • the special equipment of the present invention is characterized in that the structure of the bracket is cylindrical at both ends and prismatic at both ends, and the material is stainless steel or titanium alloy.
  • the special device of the present invention is characterized in that: the first Y-direction slider and the second Y-direction slider respectively have a first small guide rail and a second small guide rail parallel to the Y-direction guide rail, and the first small guide rail and The first small slider and the second small slider are respectively mounted on the second small guide rail, and the first small slider and the second small slider are provided with holes for fixing the nozzle.
  • a method of preparing a tissue organ using a spin stacking method characterized in that the method comprises the following steps:
  • a three-dimensional model is designed by using three-dimensional drawing software, and a mold having the lumen structure is formed by rapid prototyping, when forming the organ of the entity,
  • the mold is a cylinder with a diameter of 8 to 10 mm; the bracket is placed in the center of the mold, and the gelatin solution is added to the mold, and the temperature is lowered to 8 to 10 ° C to cure the gelatin, and the mold is opened to obtain the core of the tissue organ with the stent in the center.
  • the tissue organ is coated with a hydrogel outside the core and crosslinked/polymerized with a crosslinking agent/polymerization solution;
  • the seed cell-containing hydrogel solution and the cell-free hydrogel solution are respectively charged into the first squeeze head and the second squeeze head, the cross-linking agent/polymerization solution, the nerve cell suspension, and the endothelial cell
  • the suspension and the synthetic polymer solution are respectively charged into the fourth spray head, the second spray head, the first spray head and the third spray head, and the cell culture liquid is filled in the forming container, and the bracket is installed in the forming container Connected to the motor output shaft, the model of the tissue to be formed is introduced into the control system, the forming equipment is started, and the stacking is started.
  • the bracket rotates around the negative Y direction, and the nozzle performs the translational movement when the forming tissue is formed.
  • the first squeeze nozzle moves to the designated position to extrude the hydrogel containing the seed cells and is wound on the inner core
  • the fourth spray nozzle equipped with the crosslinking agent/polymerization liquid moves to the first
  • the cross-linking agent/polymerization solution is sprayed directly behind the squeeze nozzle to cross-link/polymerize
  • the second squeeze nozzle moves to a designated position to extrude the cell-free hydrogel Wrap around the inner core while the fourth spray nozzle moves to the rear of the second squeeze nozzle, and the first spray nozzle and the third spray nozzle respectively move to the front of the second squeeze nozzle, within the rotation Under the core
  • the newly wound hydrogel moves to the fourth spray nozzle for cross-linking/polymerization, and is washed by the culture liquid to spray the endothelial cells and the composite under the first spray nozzle and the third spray nozzle.
  • Synthetic polymer solution when forming the nerve, the second squeeze nozzle moves to the designated position to extrude the cell-free hydrogel wound on the inner core, and the fourth spray nozzle moves to the second squeeze nozzle At the rear, the second spray nozzle and the third spray nozzle respectively move to the front of the second squeeze nozzle, and under the rotating inner core, the newly wound hydrogel moves to the fourth spray nozzle to perform the intersection.
  • the tissue organ precursor containing the inner core is cultured in an incubator, the inner core is removed, and the stent is taken out, and after being cultured, a rotating body tissue is formed, and the nerve and the blood vessel penetrate through the spiral.
  • the method for preparing a tissue organ according to the present invention is characterized in that: the seed cell is a stem cell having differentiation ability or a physiologically active adult cell; and the stem cell is adipose stem cell, embryonic stem cell, blood stem cell, bone marrow stem cell, Inducible pluripotent stem cells, the adult cells are adipocytes, hepatocytes, bladder cells, lymphocytes, cardiomyocytes or kidney cells; the hydrogel is a combination of gelatin, collagen, matrigel, carrageenan, chitosan a sodium alginate or fibrinogen solution of one or more of agar, hyaluronic acid, matrigel, elastin and laminin; the hydrogel can be combined with various cell cryopreservatives, cell growth a factor, a drug, and an anticoagulant; the cell cryopreservative is one or more complexes of dimethyl sulfoxide (DMSO), glycerol, and dextrose; the cell growth factor is
  • the present invention has the following advantages and outstanding technical effects:
  • the invention combines a core-dissolving method and a 3D printing method, and can form a tissue organ having a lumen which is difficult to form by a conventional 3D printing method;
  • FIG. 1 is a schematic diagram of a three-dimensional structure of a dedicated device of the present invention.
  • Figure 3 is a schematic view showing the process of forming a tissue organ of the present invention.
  • Figure 4 is a flow chart showing the process of forming a tissue organ of the present invention.
  • Figure 5a is a cell-free hydrogel
  • Figure 5b is a hydrogel sprayed with endothelial (neural) cells
  • Figure 5c is a hydrogel sprayed with a synthetic polymer solution.
  • Figure 6 is a schematic view showing the structure of a tissue organ of the present invention.
  • Fig. 1 is a special apparatus for preparing a tissue organ by a spin stacking method provided by the present invention, the apparatus comprising a multi-nozzle assembly, a moving mechanism, a forming device, and a control system (not shown).
  • the moving mechanism is realized by a slider-guide rail (slot), including an X-direction rail 121 and an X-direction slider 120, a Y-direction rail 107 and a first Y-direction slider 111, a second Y-direction slider 112, and a Z-direction guide rail.
  • the first squeeze nozzle 113 and the second squeeze nozzle The nozzle inner diameter of 114 is 200 ⁇ m -1 mm and 50 ⁇ m -500 ⁇ m, respectively, and the four spray nozzle nozzles have a mesh diameter of 50 ⁇ m - 100 ⁇ m;
  • the forming device comprises a forming container 102, a bracket 103 and a motor 119, and the forming container 102 is mounted on Directly below the head assembly on the bottom plate 101, the detachable bracket 103 is mounted in the forming container 102 and connected to the output shaft of the motor 119, which is a solid material having a certain mechanical strength and is non-toxic to cells, such as stainless steel or Titanium alloy;
  • the control system is responsible for controlling the three-dimensional translational movement of the nozzle assembly and the rotational movement of the bracket.
  • FIG. 2 is a schematic diagram of a three-dimensional structure of a first Y-direction slider, wherein the first Y-direction slider has two holes for fixing the nozzle and a large hole for moving the nozzle, and the two sides of the large hole are provided with a small guide rail 110, the first small slider 109 can be moved along the small guide rail, and the third spray nozzle 108 driven thereon is rotated for the second spray nozzle 105 of the first spray nozzle 104, and the second Y-direction slider structure Similar to the first Y-slide.
  • FIG. 3 is a flow chart of forming a tissue organ by a spin stacking method according to the present invention, which mainly includes three main steps of preparation work before forming, formation of organ precursors, and subsequent operations.
  • Preparation before forming includes preparation of materials and preparation of inner core, preparation of 0.2-20% (w/v) hydrogel solution, 1-10% (w/v) crosslinker/polymerization solution, 0.1-10% (w/v) synthetic polymer solution, 0.2-10% (w/v) gelatin solution and cell culture solution; according to the type of tissue and organs to be formed, isolate autologous stem cells, such as adipose stem cells, from the patient , embryonic stem cells, blood stem cells, bone marrow stem cells, inducible pluripotent stem cells, or physiologically active adult cells, such as adipocytes, hepatocytes, bladder cells, lymphocytes, cardiomyocytes, or kidney cells as seed cells, and made into cell suspensions
  • the density of the seed and the seed cells is 1 ⁇ 10 3 to 1 ⁇ 10 8 /mL, and the seed cell suspension and the sterilized hydrogel solution are uniformly mixed in a volume ratio of 1 to 9:9 to 1 to prepare a mixture.
  • the cell growth factor is an endothelial cell growth factor (such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), hepatocyte growth factor (HGF), human platelet-derived growth factor (PDGF-). BB), one or more complexes of transforming growth factor ⁇ 1 (TGF- ⁇ 1).
  • the drug is an antitumor drug (such as astragalus polysaccharide, cis diaminodichloroplatinum (DDP), and mitogen (MMC), 5-fluorouracil (5-FU), anti-allergic drugs, antibiotics (such as penicillin, gibberellin, tetracycline), one or more complexes of viral vaccines.
  • VEGF vascular endothelial growth factor
  • b-FGF basic fibroblast growth factor
  • HGF hepatocyte growth factor
  • PDGF- human platelet-derived growth factor
  • BB transforming growth factor ⁇ 1
  • the drug is an antitumor
  • the anticoagulant factor is One or more complexes of heparin and paclitaxel; the solute of the synthetic polymer solution is specifically polyurethane (PU), polycaprolactone (PCL), polycarbonate, polyethylene glycol, polylactic acid-glycolic acid One or more of a copolymer (PLGA), a polyester, and a polyhydroxy acid ester, the solvent is tetraethylene glycol or 1,4-dioxane; the stent 103 on the shaped container 102 is taken out and sterilized When forming a tissue organ having a lumen, according to the structural characteristics of the lumen of the tissue organ to be formed, The three-dimensional drawing software is designed into a three-dimensional model, and a mold having the inner cavity structure is formed by rapid prototyping.
  • the bracket 103 is placed in the mold, a gelatin solution is added to the mold, and the temperature is lowered to 10 to 12 ° C to solidify the gelatin to obtain a center belt bracket.
  • FIG. 4 is a schematic view of a spin build process of the present invention, in which a hydrogel containing a seed cell and a hydrogel containing no cells are separately loaded into a first squeeze head 113 and a second squeeze head 114 in the apparatus, The cross-linking agent/polymerization solution, the nerve cell suspension and the endothelial cell suspension are respectively charged into the fourth spray head 115, the second spray head 105 and the first spray head 104, and the synthetic polymer solution is charged into the third spray.
  • the fourth spray head 115 equipped with the cross-linking agent/polymerization liquid is moved to the front of the first squeeze head 113 to spray the cross-linking agent/polymerization liquid to cross-link/polymerize it; when forming the blood vessel, The two squeeze nozzles 114 are moved to the designated position to extrude the cell-free hydrogel wound on the inner core, while the fourth spray nozzle 115 moves to the rear of the second squeeze nozzle 114, and the first spray nozzle 104 And the third spray nozzle 108 moves to the front of the second squeeze nozzle 114, respectively, and the newly wound hydrogel moves to the fourth spray nozzle 115 for crosslinking/polymerization under the rotation of the inner core.
  • the tissue organ is a revolving body or a near-rotating body, and is composed of an inner cavity 603 and a peripheral entity; the inner cavity 603 is cylindrical, spherical or surrounded by other curved surfaces. Closed surface shape;
  • the peripheral entity is wound up and accumulated by hydrogel filaments containing or not containing cells; hydrogel filaments 602 containing seed cells are entangled to form a host structure, and cell-free hydrogel filaments 603 are coated with endothelial cells and synthesized.
  • the polymer solution forms a vascular system, and the cell-free hydrogel surface coats the nerve cells and the synthetic polymer solution to form the nervous system.
  • DMSO sterilized to obtain gelatin-alginate hydrogel material
  • the degradable polycarbonate is dissolved in tetraethylene glycol to obtain a synthetic polymer solution having a mass volume concentration of 2%, and is sterilized for use
  • the cell culture solution DMEM and the 1% collagen solution are prepared.
  • a mixture of stellate cells and cardiomyocytes and a hydrogel are mixed at a volume ratio of 1:1 to obtain a hydrogel solution containing myocardial-stellate cells.
  • a three-dimensional spindle model was designed by using three-dimensional drawing software, and a mold with the inner cavity structure was prepared by rapid prototyping.
  • the sterilized stent was placed in the mold, and a gelatin solution was added to the mold to reduce The temperature is 10-12 ° C to cure the gelatin, forming a central core with a stent, and a layer of collagen solution is coated on the core of the stent, and the collagen is solidified to form a protective film insoluble in the aqueous solution;
  • the hydrogel containing the myocardial-stellate cells and DMSO and the cell-free hydrogel were separately loaded into the first squeeze head 113 and the first squeeze head 114, and the endothelial cell suspension and the nerve cell suspension were respectively.
  • the first spray nozzle 104 and the second spray nozzle 105 are installed, the calcium chloride aqueous solution is loaded into the fourth spray nozzle 115, the synthetic polymer solution is loaded into the third spray nozzle 108, and the bracket with the gelatin core is installed.
  • 100 mL of the cell culture liquid is placed in the forming container 102, and the entire apparatus is placed in an environment having a temperature of 8 to 10 ° C.
  • the atrial model is introduced into the control system, and the forming device is started. Forming.
  • the atrial precursor containing the gelatin core is placed in liquid nitrogen for long-term storage or incubator, the inner core is removed, and the stent is taken out to obtain an atrial precursor with a lumen.
  • the atrial precursor is cultured in a pulsating biological incubator.
  • the cells are connected and induced to grow, and the endothelial (neural) cells around the cell-free gel filament are induced.
  • Differentiation forms a blood vessel (nerve) with an inner diameter of about 1 mm, and after gel degradation, finally forms a blood vessel and nerve-containing atrium having autonomic contractility.
  • This atrium can be attached directly to the damaged heart to achieve the purpose of repairing regeneration.
  • BSCs human bone marrow stem cells
  • ADSCs adipose stem cells
  • ECs endothelial cells
  • nerve cells and hepatocytes culture and pass through to obtain seed cells
  • dissolving PU in tetraethylene glycol to obtain a synthetic polymer solution having a mass volume concentration of 5%, and sterilizing for use
  • the bone marrow stem cell-hepatocytes prepared above were mixed with the fibrinogen-gelatin-xanthine polysaccharide hydrogel material to obtain hydrocoagulation of hepatocyte-adipose stem cells having a density of 1 ⁇ 10 6 cells/mL of adipose stem cells and hepatocytes. gum.
  • Endothelial cells and adipose-derived stem cells were mixed with 0.1% heparin phosphate buffer (PBS) to obtain endothelium-stem stem cell suspensions each having a concentration of 5 ⁇ 10 7 /mL.
  • PBS heparin phosphate buffer
  • the nerve cells and the adipose stem cells were mixed to obtain a neuro-fat stem cell suspension having a concentration of 5 ⁇ 10 7 /mL.
  • the surface of the stent after sterilization is coated with a gelatin having a thickness of about 5 mm, and the temperature is lowered to 10 to 12 ° C to form an inner core, and the hydrogel is coated on the outer core of the stent before the core is cross-linked to form an insoluble layer.
  • a protective film for the culture solution is coated with a gelatin having a thickness of about 5 mm, and the temperature is lowered to 10 to 12 ° C to form an inner core, and the hydrogel is coated on the outer core of the stent before the core is cross-linked to form an insoluble layer.
  • the fibrinogen-gelatin-xanthine polysaccharide hydrogel containing hepatocyte-bone marrow stem cells and the cell-free hydrogel are respectively loaded into the first squeeze head 113 and the first squeeze head 114, endothelium-fat stem cells
  • the heparin suspension and the neuro-fat stem cell suspension are respectively filled into the first spray nozzle 104 and the second spray nozzle 105, the thrombin polymerization liquid is charged into the fourth spray nozzle 115, and the synthetic polymer is charged into the third spray type.
  • a stent with a gelatin core was mounted in the forming vessel 102 and connected to the motor output shaft, and 100 mL of the cell culture solution was placed in the forming vessel. The entire device was placed in an environment at a temperature of 10 ° C, and the liver precursor model was introduced to start the forming device to start forming.
  • the liver precursor containing the gelatin core is placed in a constant temperature environment of about 37 ° C, the gelatin core is "melted", and the stent is taken out to obtain the liver precursor.
  • the liver precursor is cultured in an incubator. During the culture, the cells establish a connection and proliferate, and the endothelial (neural) cells and adipose stem cells around the cell-free gel filaments are induced to differentiate into blood vessels having an inner diameter of about 1 mm ( Nerve), after the gel degrades, eventually forms the liver.
  • the endothelial (neural) cells and adipose stem cells around the cell-free gel filaments are induced to differentiate into blood vessels having an inner diameter of about 1 mm ( Nerve), after the gel degrades, eventually forms the liver.
  • the ellipsoidal structure was designed by three-dimensional drawing software, and the mold with the inner cavity structure was fabricated by rapid prototyping.
  • the sterilized stent is placed in a mold, an appropriate amount of gelatin solution is added to the mold, the temperature is lowered to 10 to 12 ° C to cure the gelatin, and the inner core of the tissue organ with the stent is formed, and the core is coated with the stent in the liver.
  • the hydrogel is coated and crosslinked to form a protective film that is insoluble in the culture solution.
  • the bladder precursor containing the gelatin core is cultured in an incubator, the inner core is removed, and the stent is taken out to obtain a bladder precursor with a lumen.
  • the bladder precursor is placed in an incubator for cultivation.
  • the cells establish a connection and proliferate, and the endothelial (neural) cells and adipose stem cells around the cell-free gel filaments are induced to differentiate into blood vessels (nerves).
  • blood vessels nerves and blood vessels is finally formed.
  • the hydrogel containing the renal cell-blood stem cells and the cell-free hydrogel are separately loaded into the first squeeze head 113 and the second squeeze head 114, the endothelial cell-blood stem cell solution and the nerve cell-blood stem cell
  • the solution is separately charged into the first spray head 104 and the second spray head 105, the calcium chloride crosslinker/polymerization liquid is charged into the fourth spray head 115, and the synthetic polymer is charged into the third spray head 108.
  • a stent with a gelatin core was mounted in the forming vessel 102 and connected to the motor output shaft, and 100 mL of the cell culture solution was placed in the forming vessel.
  • the entire apparatus was placed in an environment having a temperature of 8 to 10 ° C, and after the kidney model was introduced, the forming apparatus was started, and the spin-up forming was started.
  • the kidney precursor containing the inner core of the gelatin film is cultured in an incubator, the inner core of the gelatin film is "melted", and the stent is taken out to obtain a kidney precursor.
  • the kidney precursor is cultured in an incubator. During the culture, the cells establish a connection and proliferate, and the endothelial (neural) cells and blood stem cells around the cell-free gel filament are induced to differentiate into blood vessels having an inner diameter of about 1 mm. (Nerve), after gel degradation, eventually forms a kidney with blood vessels and nerves.
  • ADSC Human adipose stem cells
  • EC endothelial cells
  • nerve cells and lymphocytes were extracted and cultured and passaged to obtain cell materials; 1 g of sodium alginate and 2 g of gelatin were placed in 100 mL of culture solution to completely dissolve them, and sterilization was carried out.
  • Sodium alginate-gelatin hydrogel material preparing 0.05g/ml calcium chloride aqueous solution as its crosslinking agent/polymerization; taking 2g gelatin in 100mL culture solution to completely dissolve it, and sterilizing to obtain gelatin solution;
  • the synthetic polymer PU is dissolved in tetraethylene glycol to obtain a synthetic polymer solution having a mass volume concentration of 5%, and is sterilized for use;
  • the adipose stem cells prepared above were mixed with a sodium alginate-gelatin hydrogel material to obtain a hydrogel containing adipose stem cells having a fat stem cell density of 1 ⁇ 10 6 /mL.
  • 1 ⁇ M insulin, 1 ⁇ M dexamethasone, 50 u/mL aprotinin and 0.5 mmol/L isobutylmethylated xanthine derivative were added thereto.
  • the nerve cells and the adipose stem cells were mixed to obtain a nerve cell-fat stem cell suspension having a concentration of 3 ⁇ 10 8 /mL.
  • 50 ng/mL endothelial growth factor (VEGF), 3 ng/mL TGF- ⁇ 1 growth factor, and 10 ng/mL fibroblast growth factor (b-FGF) were added to the adipose stem cell suspension.
  • VEGF endothelial growth factor
  • b-FGF fibroblast growth factor

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Abstract

一种利用旋转堆积法制备组织器官的专用设备，包括支撑装置、多喷头组件、运动机构、成形装置以及控制系统。利用该设备制备组织器官的方法基于溶芯技术和细胞组装技术，将各种细胞生长因子、抑制因子或药物复合到可交联/聚合的水凝胶材料中，从喷头中挤出的丝状水凝胶经交联/聚合、合成高分子复合、培养液洗涤处理后缠绕堆积在旋转内芯上，含不同类型种子细胞的水凝胶和粘附内皮细胞、神经细胞的水凝胶分别形成主体部分和血管、神经系统，成形完毕后溶除内芯并培养一段时间后得到具有螺旋状血管和神经的组织器官。该制备方法简单且易于成形血管和神经系统，特别适用于成形传统组织工程难以成形的具有内腔的组织器官。

Description

利用旋转堆积法制备组织器官的方法及专用设备 技术领域
本发明涉及利用旋转堆积法制备组织器官的方法及专用设备，属于组织工程领域。
背景技术
人体病损组织和器官的修复和替代是本世纪令人瞩目的科学前沿，也是一项亟需解决的科学难题。由于活体组织器官移植存在供体短缺以及免疫排斥风险大等固有弊端，难以成为解决该问题的有效途径。据此，以提高此类疾患治疗水平为目的的组织工程(Tissue Engineering)应运而生，组织工程旨在体外构建细胞和材料的三维结构体，通过适当的培养和训练后发展成具有和天然组织类似结构和功能的可移植替代物。近年来，组织工程一直是国内外研究的热点，发展迅速并取得了诸多成果，但就其产品应用领域而言，目前主要集中在结构相对简单的骨组织工程和皮肤组织工程等少数几个领域。究其原因，很重要的一点就是，现有的组织工程结构体的体外构建方法(如电纺丝、模铸法等)难以构建具有天然组织类似的复杂的血管网络结构，致使结构体内部的细胞存活率低，因此难以实现大块组织的构建。
传统的组织工程遵循“细胞-支架复合”的“自上而下”的构建思路，即先成形支架，再把细胞种植到支架上。但是，当支架尺寸和复杂度提高到一定程度时，支架成形和细胞复合的难度将大大增加。细胞三维受控组装技术(Wang X H,Yan Y N,Zhang R J.Rapid prototyping as a tool for manufacturing bio-artificial livers.Trends in Biotechnology,2007,25(11):505-513.)，即细胞3D打印技术，遵循“支架-细胞”一体化构建的思路。在细胞打印过程中，细胞(或细胞聚集体)与溶胶(水凝胶的前驱体)同时置于或者含细胞的培养液单独置于打印机的喷头中，由计算机控制含细胞液滴的沉积位置，在指定的位置逐点打印，在打印完一层的基础上继续打印另一层，层层叠加形成三维多细胞‐凝胶体系。细胞打印技术的特点是可以保证细胞密度和分布的均匀性，并且可以实现多种细胞的共同组装和定点排布。但是氧气和营养物质的有效输送问题，即“血管化问题”仍然是该方法构建可移植复杂组织器官的瓶颈问题。
现有的通过细胞3D打印成形的组织器官都是按照一层一层的平面堆积而成的，因其成形的固有特点，微通道(血管)的成形依赖于成形精度及材料的性质。而且对于具有内腔结构 组织器官的成形，依靠平面堆积的细胞打印技术在其成形过程中不可避免地需要增加支架或者转动成形台或喷头方位，大大增加了设计和成形的难度，有时甚至还无能为力。
中国专利文献CN1654028提出一种利用溶芯法制造管网状支架的方法，先利用水溶性、无生物毒性的材料制造支架的内芯，然后将生物相容性材料涂覆在支架内芯上，经风干后用蒸馏水溶除支架内芯。该方法只能成形结构较简单的管网状支架，对于具有复杂血管的组织器官的成形无能为力。
发明内容
本发明的目的在于克服传统的组织工程中难以构建具有复杂血管和神经结构的空腔类组织器官，提供一种基于旋转堆积法制备组织器官的方法及专用设备。该方法在细胞受控组装技术和溶芯技术的基础上，目的在于提供一种新的组织器官的制备方法，便于制备具有螺旋网状血管和神经的组织器官，特别适用于制备具有内腔结构的组织器官。
本发明的技术方案如下：
利用旋转堆积法制备组织器官的工艺方法，其特征在于本发明的专用设备包括支撑装置、多喷头组件、运动机构、成形装置以及控制系统；所述支撑装置包括支撑腿和底板；所述运动机构采用导轨‐滑块机构，包括X向导轨、X向滑块、Y向导轨、第一Y向滑块、第二Y向滑块、Z向导轨、Z向滑块，所述X向导轨设在底板上，Z向导轨固接在X向滑块上，Y向导轨固接在Z向滑块上；所述多喷头组件至少含有六个喷头，包括至少两个挤压式喷头和四个喷洒式喷头，两个挤压式喷头的喷嘴内径分别为200μm～1mm和50μm～500μm，喷洒式喷头喷嘴的网孔直径为50μm～100μm，喷头分两组分别安装在第一Y向滑块和第二Y向滑块上；所述成形装置包括成形容器、支架和电机，成形容器安装在底板上并处于喷头组件的下部，支架安装在成形容器内并与电机输出轴相连。
本发明所述专用设备其特征在于：所述支架的结构为两端呈圆柱状、中间为棱柱状，材料为不锈钢或者钛合金。
本发明所述专用设备其特征在于：所述第一Y向滑块和第二Y向滑块上分别开有与Y向导轨平行的第一小导轨和第二小导轨，第一小导轨和第二小导轨上分别安装第一小滑块)和第二小滑块，第一小滑块和第二小滑块上开有可固定喷头的孔。
利用旋转堆积法制备组织器官的方法，其特征在于该方法包括以下步骤：
1)材料制备：
制备0.2～20％(w/v)水凝胶溶液、1～10％(w/v)交联剂/聚合液、0.1～10％(w/v)合 成高分子溶液、0.2～10％(w/v)明胶溶液和细胞培养液；分离或者诱导种子细胞，种子细胞密度为1×103～1×106个/mL，将种子细胞与水凝胶溶液按1～9：9～1体积比混合制成含种子细胞的水凝胶溶液；分离或者诱导神经细胞和内皮细胞，并制成细胞悬液，神经细胞悬液和内皮细胞悬液密度均为1×103～1×108个/mL；
2)内芯制备
当制备具有内腔的组织器官时，根据所要制备组织器官的内腔形状，用三维绘图软件设计其三维模型，用快速成形法制作具有此内腔结构的模具，当成形实体的组织器官时，模具是直径厚8～10mm的圆柱；将支架放入模具中央，往模具中加入明胶溶液后降低温度至8～10℃使明胶固化，打开模具得到中心带支架的组织器官内芯，在带支架的组织器官内芯外涂覆水凝胶，并用交联剂/聚合液进行交联/聚合；
3)成形
将含种子细胞的水凝胶溶液和不含细胞的水凝胶溶液分别装入第一挤压式喷头和第二挤压式喷头中，交联剂/聚合液、神经细胞悬液、内皮细胞悬液以及合成高分子溶液分别装入第四喷洒式喷头、第二喷洒式喷头、第一喷洒式喷头和第三喷洒式喷头中，在成形容器内装入细胞培养液，将支架安装在成形容器内并与电机输出轴相连，将待成形组织器官的模型导入控制系统后，启动成形设备，开始堆积成形；在成形过程中，支架绕Y负向做旋转运动，喷头做平移运动，当成形组织器官的主体部分时，第一挤压式喷头运动到指定位置挤出含种子细胞的水凝胶缠绕在内芯上，同时装有交联剂/聚合液的第四喷洒式喷头运动到第一挤压式喷头的正后方喷洒出交联剂/聚合液对其进行交联/聚合；当成形血管时，第二挤压式喷头运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头运动到第二挤压式喷头正后方，第一喷洒式喷头和第三喷洒式喷头分别运动到第二挤压式喷头正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头下进行交联/聚合，经过培养液进行洗涤，至第一喷洒式喷头、第三喷洒式喷头下进行喷洒内皮细胞和复合合成高分子溶液；当成形神经时，第二挤压式喷头运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头运动到第二挤压式喷头正后方，第二喷洒式喷头和第三喷洒式喷头分别运动到第二挤压式喷头正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头下进行交联/聚合，经过培养液进行洗涤，至第二喷洒式喷头、第三喷洒式喷头下进行喷洒神经细胞和复合合成高分子；如此反复缠绕堆积直至形成含内芯的组织器官前体；
4)后续处理
将含内芯的组织器官前体置于恒温箱中培养，溶除内芯后取出支架，经培养后形成回转体状组织器官，神经和血管以螺旋状贯穿其中。
本发明所述制备组织器官的方法，其特征在于：所述的种子细胞为具有分化能力的干细胞或具有生理活性的成体细胞；所述的干细胞为脂肪干细胞、胚胎干细胞、血液干细胞、骨髓干细胞、诱导型多功能干细胞，所述成体细胞为脂肪细胞、肝细胞、膀胱细胞、淋巴细胞、心肌细胞或肾细胞；所述水凝胶为复合了明胶、胶原、基质胶、卡拉胶、壳聚糖、琼脂、透明质酸、基质胶、弹性蛋白和层粘素中一种或多种的海藻酸钠或纤维蛋白原溶液；所述的水凝胶中可复合各种细胞冻存剂、细胞生长因子、药物和抗凝血剂；细胞冻存剂为二甲基亚砜(DMSO)、甘油和右旋糖中的一种或多种复合物；所述的细胞生长因子为血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(b-FGF)、肝细胞生长因子(HGF)、人血小板衍化生长因子(PDGF-BB)和转化生长因子β1(TGF-β1)中的一种或多种复合物；所述的药物为抗肿瘤药物，该抗肿瘤药物为黄芪多糖、顺式二氨基二氯络铂(DDP)、丝裂霉素(MMC)，5-氟尿嘧啶(5-FU)、抗过敏药物、抗生素、病毒疫苗中的一种或多种复合物；所述的抗凝血因子为肝素、紫杉醇中的一种或多种复合物；所述合成高分子溶液的溶质为聚氨酯、聚己内酯、聚碳酸酯、聚乙二醇、聚乳酸-羟基乙酸共聚物、聚酯和聚羟基酸酯中的一种或者多种，溶剂为四乙二醇或1,4-二氧六环。
本发明与现有技术相比，有以下优点及突出性的技术效果：
①本发明将溶芯法和3D打印方法结合起来，能成形传统3D打印方法难以成形的具有内腔的组织器官；
②多喷头的使用可实现多种细胞非均质成形以及表面复合处理，满足天然组织器官对多种细胞的要求；
③在水凝胶表面喷洒内皮(神经)细胞的血管(神经)形成方式能成形具有复杂结构的血管(神经)，合成高分子溶液形成的薄膜提高了水凝胶表面粘附的内皮(神经)细胞数目，有效促进血管(神经)的形成；
④成形过程中细胞培养液既能为细胞提供营养物质又可以洗涤凝胶表面的交联剂/聚合液及合成高分子中的有机溶剂，减小其对细胞的损伤。
附图说明
图1为本发明的专用设备三维结构简图。
图2为第一Y向滑块的三维结构简图。
图3为本发明的组织器官成形过程示意图。
图4为本发明的组织器官成形工艺流程图。
图5a为不含细胞的水凝胶；图5b为喷上内皮(神经)细胞后的水凝胶；图5c为喷洒合成高分子溶液后的水凝胶。
图6为本发明的组织器官的结构示意图。
图中：101‐底板；102‐成形容器；103‐支架；104‐第一喷洒式喷头；105‐第二喷洒式喷头；106‐Z向滑块；107‐Y向导轨；108‐第三喷洒式喷头；109‐第一小滑块；110‐第一小导轨；111‐第一Y向滑块；112‐第二Y向滑块；113‐第一挤压式喷头；114‐第二挤压式喷头；115‐第四喷洒式喷头；116‐第二小滑块；117‐第二小导轨；118‐Z向导轨；119‐旋转电机；120‐X向滑块；121‐X向导轨；401‐内芯；402‐喷头组件；403‐材料；404‐水凝胶丝；405‐培养液；601‐主体部分；602‐血管(神经)；603‐内腔。
具体实施方式
下面结合附图和实施例对本发明进一步说明。
图1是本发明提供的利用旋转堆积法制备组织器官的专用设备，该设备包括多喷头组件、运动机构、成形装置以及控制系统(未画)。所述运动机构由滑块—导轨(槽)实现，包括X向导轨121和X向滑块120、Y向导轨107和第一Y向滑块111、第二Y向滑块112、Z向导轨118和Z向滑块106以及第一小滑块109、第二小滑块116和第一小导轨110、第二小导轨117；所述X向导轨121设在底板101上；所述Z向导轨118固接在X向滑块120上；所述Y向导轨107固接在Z向滑块106上；所述第一小导轨110和第二小导轨117分别安装在第一Y向滑块111和第二Y向滑块112上；所述多喷头组件至少含有六个喷头，包括至少两个挤压式喷头和四个喷洒式喷头，分两组分别固定在两个平行的Y向滑块上，第一喷洒式喷头104、第二喷洒式喷头105和第三喷洒式喷头108安装在第一Y向滑块111上，其中第一喷洒式喷头104和第二喷洒式喷头105安装在第一Y向滑块111上的喷头固定槽中，第三喷洒式喷头108安装在第一小滑块109的喷头固定槽上；第四喷洒式喷头115和至少两个挤压式喷头，第一挤压式喷头113和第二挤压式喷头114，安装在第二Y向滑块112上，其中第一挤压式喷头113和第二挤压式喷头114固定在第二Y向滑块112的喷头固定槽上，第四喷洒式喷头115安装在第二小滑块116的喷头固定槽上，第一挤压式喷头113和第二挤压式喷头114的喷嘴内径分别为200μm‐1mm和50μm‐500μm，四个喷洒式喷头喷嘴的网孔直径为50μm‐100μm；所述成形装置包括成形容器102、支架103以及电机119，成形容器102安装在 底板101上喷头组件的正下方，可拆卸的支架103安装在成形容器102内并与电机119的输出轴相连，所述支架103为具有一定力学强度的对细胞无毒性的固体材料，如不锈钢或者钛合金；所述控制系统负责控制喷头组件的三维平移运动和支架的旋转运动。
图2为第一Y向滑块的三维结构简图，所述第一Y向滑块上开有两个用来固定喷头的孔和一个能让喷头运动的大孔，大孔两边设有第一小导轨110，第一小滑块109可沿小导轨运动，带动其上的第三喷洒式喷头108轮流为第一喷洒式喷头104第二喷洒式喷头105工作，第二Y向滑块结构与第一Y向滑块类似。
图3为本发明的利用旋转堆积法成形组织器官的流程图，主要包括成形前的准备工作、组织器官前体成形以及后续操作等三大主要步骤。
成形前的准备工作包括材料的制备和内芯的制作，制备0.2～20％(w/v)水凝胶溶液、1～10％(w/v)交联剂/聚合液、0.1～10％(w/v)合成高分子溶液、0.2～10％(w/v)的明胶溶液和细胞培养液；根据所要成形的组织器官的类型，从病人身上分离具有分化能力的自体干细胞，如脂肪干细胞、胚胎干细胞、血液干细胞、骨髓干细胞、诱导型多功能干细胞，或具有生理活性成体细胞，如脂肪细胞、肝细胞、膀胱细胞、淋巴细胞、心肌细胞或肾细胞作为种子细胞，并制成细胞悬液，种子细胞密度为1×103～1×108个/mL，将种子细胞悬液与灭菌后的水凝胶溶液按体积比为1～9：9～1均匀混合，制备成含种子细胞的水凝胶混合物；所述水凝胶为可复合明胶、胶原、基质胶、卡拉胶、壳聚糖、琼脂、透明质酸、基质胶、弹性蛋白、层粘素中一种或多种的海藻酸钠或纤维蛋白原溶液，所述的水凝胶中可复合各种细胞冻存剂、细胞生长因子、药物、抗凝血剂。细胞冻存剂为二甲基亚砜(DMSO)、甘油、右旋糖中的一种或多种复合物。所述的细胞生长因子为内皮细胞生长因子(如血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(b-FGF)、肝细胞生长因子(HGF)、人血小板衍化生长因子(PDGF-BB)、转化生长因子β1(TGF-β1)中的一种或多种复合物。所述的药物为抗肿瘤药物(如黄芪多糖、顺式二氨基二氯络铂(DDP)、丝裂霉素(MMC)，5-氟尿嘧啶(5-FU)、抗过敏药物、抗生素(如青霉素、赤霉素、四环素)、病毒疫苗中的一种或多种复合物。所述的抗凝血因子为肝素、紫杉醇中的一种或多种复合物；所述合成高分子溶液的溶质具体为聚氨酯(PU)、聚己内酯(PCL)、聚碳酸酯、聚乙二醇、聚乳酸‐羟基乙酸共聚物(PLGA)、聚酯和聚羟基酸酯中的一种或者多种，溶剂为四乙二醇或1,4‐二氧六环；取出成形容器102上的支架103并进行灭菌处理，当成形具有内腔的组织器官时，根据所要成形的组织器官内腔的结构特点，用三维绘图软件设计其三维模型，用快速成形法制作具有此内腔结构的模具，将支 架103立入模具中，往模具中加入明胶溶液，降低温度至10～12℃使明胶固化，得到中心带支架的组织器官内芯；当成形实体组织器官时，内芯为厚5～6mm的圆柱套筒；然后在带支架的组织器官内芯外涂覆一层水凝胶，并用交联剂/聚合液进行交联/聚合。
图4为本发明的旋转堆积成形过程示意图，将含种子细胞的水凝胶和不含细胞的水凝胶分别装入设备中第一挤压式喷头113和第二挤压式喷头114中，交联剂/聚合液、神经细胞悬液和内皮细胞悬液分别装入第四喷洒式喷头115、第二喷洒式喷头105和第一喷洒式喷头104中，合成高分子溶液装入第三喷洒式喷头喷头108中，将支架103安装在成形容器102中并在成形容器102内装入细胞培养液405；将待成形组织器官的模型导入控制系统后，启动成形设备，开始堆积成形，在成形过程中，支架103绕Y负向做旋转运动，喷头做平移运动，当成形组织器官主体部分时，第一挤压式喷头113运动到指定位置挤出含种子细胞的水凝胶缠绕在内芯上，同时装有交联剂/聚合液的第四喷洒式喷头115运动到第一挤压式喷头113的正后方喷洒出交联剂/聚合液对其进行交联/聚合；当成形血管时，第二挤压式喷头114运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头115运动到第二挤压式喷头114正后方，第一喷洒式喷头104和第三喷洒式喷头108分别运动到第二挤压式喷头114正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头115下进行交联/聚合，经过培养液进行洗涤，经过第一喷洒式喷头104、第三喷洒式喷头108下进行喷洒内皮细胞和复合合成高分子；当成形神经纤维束时，第二挤压式喷头114运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头115运动到第一挤压式喷头113正后方，第二喷洒式喷头105和第三喷洒式喷头108分别运动到第二挤压式喷头114正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头115下进行交联/聚合，经过培养液进行洗涤，经过第二喷洒式喷头105、第三喷洒式喷头108下进行喷洒神经细胞和复合合成高分子，如此反复缠绕堆积，直至成形所需的组织器官。
图5a为不含细胞的水凝胶；图5b为喷上内皮(神经)细胞后的水凝胶；图5c为喷洒合成高分子溶液后的水凝胶；成形血管(神经)时，从喷头中喷出的不含细胞的水凝胶丝经交联/聚合和洗涤后，依次被喷上一层内皮(神经)细胞和合成高分子溶液，形成凝胶‐细胞‐薄膜结构，在后续培养过程中，水凝胶逐渐水解，内皮细胞或者神经细胞生长并相互联系，最终形成血管和神经结构。
图6为本发明提供的组织器官的结构示意图，所述组织器官为回转体或近回转体，由内腔603和外围实体组成；所述内腔603为圆柱状、球状或者由其他曲面围成的闭合曲面形状； 所述外围实体由含或者不含细胞的水凝胶丝缠绕堆积而成；含有种子细胞的水凝胶丝602缠绕形成主体结构，不含细胞的水凝胶丝603表面涂覆内皮细胞和合成高分子溶液形成血管系统，不含细胞的水凝胶表面涂覆神经细胞和合成高分子溶液形成神经系统。
下面举出几个具体的实施例，以进一步理解本发明：
实施例1心房的制备
(1)材料制备
提取脂肪干细胞(ADSCs)、内皮细胞(ECs)、神经细胞、星状细胞和心肌细胞(CMCs)，培养传代，得到细胞材料，内皮细胞和神经细胞浓度均为8×107个/mL，星状细胞和心肌细胞密度为2×106个/mL；取2g海藻酸钠和7g明胶置于100mL培养液中使其完全溶解，在该混合液中加入质量体积浓度3％的细胞冻存液DMSO，灭菌处理得到明胶‐海藻酸钠水凝胶材料；取4g明胶置于100mL培养液中使其完全溶解，灭菌处理得到明胶溶液；制备0.05g/ml的氯化钙水溶液作为其交联剂/聚合液；将可降解聚碳酸酯溶于四乙二醇中得到质量体积浓度为2％的合成高分子溶液，灭菌处理待用；制备细胞培养液DMEM和1％胶原溶液。将星状细胞和心肌细胞的混合液与水凝胶以体积比为1:1混合得到含心肌‐星状细胞的水凝胶溶液。
(2)内芯制备
根据心房内腔的形状结构特点，用三维绘图软件设计三维纺锤状模型，用快速成形法制作具有此内腔结构的模具，将灭菌后的支架立入模具中，往模具中加入明胶溶液，降低温度至10～12℃使明胶固化，形成中心带支架的心房内芯，并在带支架的心房内芯外涂覆一层胶原溶液，并使胶原固化形成一层不溶于水溶液的保护膜；
(3)成形
将含心肌‐星状细胞和DMSO的水凝胶和不含细胞的水凝胶分别装入第一挤压式喷头113和第一挤压式喷头114，内皮细胞悬液和神经细胞悬液分别装入第一喷洒式喷头104、第二喷洒式喷头105，氯化钙水溶液装入第四喷洒式喷头115，合成高分子溶液装入第三喷洒式喷头108，将带明胶内芯的支架安装在成形容器102内并与电机输出轴相连，在成形容器102内装入100mL的细胞培养液，整个装置置于温度为8～10℃的环境中，将心房模型导入控制系统后启动成形装置，开始成形。
(4)溶芯
成形完毕后，将含明胶内芯的心房前体置于液氮中长期保存或恒温箱中培养，融除内芯，取出支架，即得到带内腔的心房前体。
(5)后期培养
将心房前体至于脉动生物培养器中进行培养，培养过程中，在应力场的多重作用下，促使细胞间建立连接并诱导生长，在不含细胞的凝胶丝周围的内皮(神经)细胞诱导分化形成内径约为1mm的血管(神经)，在凝胶降解后，最终形成具有自主收缩能力的含有血管和神经的心房。此心房可直接贴附在病损心脏上，达到修复再生的目的。
实施例2肝脏前体的制备
(1)材料制备
提取人体骨髓干细胞(BSCs)、脂肪干细胞(ADSCs)、内皮细胞(ECs)、神经细胞和肝细胞，培养传代，得到种子细胞；取1纤维蛋白原和7g明胶置于100mL培养液中使其完全溶解，其中加入0.1％的抗肿瘤药物黄芪多糖，灭菌处理得到纤维蛋白原‐明胶‐黄芪多糖水凝胶材料；取4g明胶置于100mL培养液中使其完全溶解，灭菌处理得到明胶溶液；制备0.05g/ml的凝血酶PBS聚合液；将PU溶于四乙二醇中得到质量体积浓度为5％的合成高分子溶液，灭菌处理待用；
将上述制备的骨髓干细胞‐肝细胞与纤维蛋白原‐明胶‐黄芪多糖水凝胶材料混合得到脂肪干细胞和肝细胞的密度均为1×106个/mL的含肝细胞‐脂肪干细胞的水凝胶。内皮细胞、脂肪干细胞与0.1％肝素磷酸缓冲液(PBS)混合得到浓度均为5×107个/mL的内皮‐脂肪干细胞悬液。神经细胞和脂肪干细胞混合得到浓度均为5×107个/mL的神经‐脂肪干细胞悬液。
(2)内芯制备
在灭菌后的支架表面涂一层厚度约为5mm的明胶，降低温度至10～12℃固化形成内芯，在带支架的肝脏前体内芯外涂覆水凝胶并交联聚合形成一层不溶于培养液的保护膜。
(3)成形
将含肝细胞‐骨髓干细胞的纤维蛋白原‐明胶‐黄芪多糖水凝胶和不含细胞的水凝胶分别装入第一挤压式喷头113和第一挤压式喷头114，内皮‐脂肪干细胞‐肝素悬液和神经‐脂肪干细胞悬液分别装入第一喷洒式喷头104和第二喷洒式喷头105，凝血酶聚合液装入第四喷洒式喷头115，合成高分子装入第三喷洒式喷头108。将带明胶内芯的支架安装在成形容器102内并与电机输出轴相连，并在成形容器内装入100mL的细胞培养液。整个装置置于温度为10℃的环境中，导入肝脏前体模型后启动成形装置开始成形。
(4)溶芯
成形完毕后，将含明胶内芯的肝脏前体置于约37℃的恒温环境中，使明胶内芯“融化”，取出支架，即得到肝脏前体。
(5)后期培养
将肝脏前体置于培养箱中培养，培养过程中，细胞间建立连接并增殖生长，不含细胞的凝胶丝周围的内皮(神经)细胞和脂肪干细胞诱导分化形成内径约为1mm的血管(神经)，在凝胶降解后，最终形成肝脏。
实施例3膀胱前体的制备
(1)材料制备
提取人体脂肪干细胞(ADSC)、内皮细胞(EC)、神经细胞和膀胱细胞，培养传代，得到细胞材料；取1g海藻酸钠、1g壳聚糖和1g明胶置于100mL培养液中使其完全溶解，灭菌处理得到海藻酸钠‐壳聚糖‐明胶水凝胶材料；制备0.05g/ml的氯化钙水溶液作为其交联剂；取2g葡萄糖和2g右旋糖置于100mL培养液中使其完全溶解，灭菌处理得到葡萄糖‐右旋糖溶液；将PLGA溶于四乙二醇中得到体积浓度为5％的溶液，灭菌处理待用；
将上述制备的脂肪干细胞‐膀胱细胞与海藻酸钠‐壳聚糖‐明胶水凝胶材料混合得到脂肪干细胞和膀胱细胞的密度均为1×106个/mL的含膀胱细胞‐脂肪干细胞的水凝胶；内皮细胞和脂肪干细胞混合得到浓度均为5×107个/mL的内皮‐脂肪干细胞悬液；神经细胞和脂肪干细胞混合得到浓度均为5×107个/mL的神经细胞‐脂肪干细胞悬液。
(2)内芯制备
根据膀胱内腔的形状结构特点，用三维绘图软件设计椭球状结构，用快速成形法制作具有此内腔结构的模具。将灭菌后的支架立入模具中，往模具中加入适量的明胶溶液，降低温度至10～12℃使明胶固化，形成中心带支架的组织器官内芯，在带支架的肝脏前体内芯外涂覆水凝胶并交联聚合形成一层不溶于培养液的保护膜。
(3)成形
将含膀胱细胞‐脂肪干细胞的海藻酸钠‐壳聚糖‐明胶水凝胶和不含细胞的水凝胶分别装入第一挤压式喷头113和第一挤压式喷头114，内皮细胞‐脂肪干细胞悬液和神经细胞‐脂肪干细胞悬液分别装入第一喷洒式喷头104和第二喷洒式喷头105，氯化钙交联剂装入第四喷洒式喷头115，合成高分子装入第三喷洒式喷头108。将带明胶内芯的支架安装在成形容器102内并与电机输出轴相连，并在成形容器内装入100mL的细胞培养液。整个装置置于温度为8～10℃的环境中，在计算机中导入膀胱模型后启动成形装置，开始成形。
(4)溶芯
成形完毕后，将含明胶内芯的膀胱前体置于恒温箱中培养，去除内芯，取出支架，即得到带内腔的膀胱前体。
(5)后期培养
将膀胱前体置于恒温箱中进行培养，培养过程中，细胞间建立连接并增殖生长，在不含细胞的凝胶丝周围的内皮(神经)细胞和脂肪干细胞诱导分化形成血管(神经)，在凝胶降解后，最终形成具有神经和血管的膀胱。
实施例4肾前体的制备
(1)材料制备
提取人体血液干细胞、内皮细胞(EC)、神经细胞和肾细胞，培养传代，得到细胞材料；取1g海藻酸钠和1g透明质酸置于100mL培养液中使其完全溶解，灭菌处理得到海藻酸钠‐透明质酸水凝胶材料；制备0.05g/ml的氯化钙水溶液作为其交联剂；取4g明胶置于100mL培养液中使其完全溶解，灭菌处理得到明胶溶液；将合成高分子PU溶于四乙二醇中得到质量体积浓度为5％的合成高分子溶液，灭菌处理待用；
将上述制备的血液干细胞‐肾细胞与海藻酸钠‐透明质酸水凝胶材料混合得到血液干细胞和肾细胞的密度均为1×106个/mL的含肾细胞‐血液干细胞的水凝胶。内皮细胞和血液干细胞混合得到浓度均为4×107个/mL的内皮细胞血液干细胞悬液。神经细胞和血液干细胞混合得到浓度均为4×107个/mL的神经细胞‐血液干细胞悬液。
(2)内芯制备
在支架表面涂一层厚约5mm的明胶，降低温度至10～12℃固化形成内芯，在带支架的肝脏前体内芯外涂覆水凝胶并交联聚合形成一层不溶于培养液的保护膜。
(3)成形
将含肾细胞‐血液干细胞的水凝胶和不含细胞的水凝胶分别装入第一挤压式喷头113和第二挤压式喷头114，内皮细胞‐血液干细胞溶液和神经细胞‐血液干细胞溶液分别装入第一喷洒式喷头104和第二喷洒式喷头105，氯化钙交联剂/聚合液装入第四喷洒式喷头115，合成高分子装入第三喷洒式喷头108。将带明胶内芯的支架安装在成形容器102内并与电机输出轴相连，并在成形容器内装入100mL的细胞培养液。整个装置置于温度为8～10℃的环境中，导入肾模型后启动成形装置，开始旋转堆积成形。
(4)溶芯
成形完毕后，将含明胶薄膜内芯的肾脏前体置于恒温箱中培养，使明胶薄膜内芯“融化”，取出支架，即得到肾脏前体。
(5)后期培养
将肾前体置于培养箱中培养，培养过程中，细胞间建立连接并增殖生长，在不含细胞的凝胶丝周围的内皮(神经)细胞和血液干细胞诱导分化形成内径约为1mm的血管(神经)，在凝胶降解后，最终形成具有血管和神经的肾脏。
实施例5人工乳房的制备
(1)材料制备
提取人体脂肪干细胞(ADSC)、内皮细胞(EC)、神经细胞和淋巴细胞，培养传代，得到细胞材料；取1g海藻酸钠和2g明胶置于100mL培养液中使其完全溶解，灭菌处理得到海藻酸钠‐明胶水凝胶材料；制备0.05g/ml的氯化钙水溶液作为其交联剂/聚合；取2g明胶置于100mL培养液中使其完全溶解，灭菌处理得到明胶溶液；将合成高分子PU溶于四乙二醇中得到质量体积浓度为5％的合成高分子溶液，灭菌处理待用；
将上述制备的脂肪干细胞与海藻酸钠‐明胶水凝胶材料混合得到脂肪干细胞密度均为1×106个/mL的含脂肪干细胞的水凝胶。其中加入1μM胰岛素,1μM地塞米松,50u/mL抑肽酶和0.5mmol/L异丁基甲基化黄嘌呤衍生物。神经细胞和脂肪干细胞混合得到浓度均为3×108个/mL的神经细胞‐脂肪干细胞悬液。脂肪干细胞悬浮液中加入50ng/mL内皮生长因子(VEGF)、3ng/mL TGF‐β1生长因子、10ng/mL成纤维细胞生长因子(b‐FGF)。
(2)内芯制备
在支架表面涂一层厚约5mm的明胶，降低温度至10～12℃固化形成内芯，在带支架的肝脏前体内芯外涂覆水凝胶并交联聚合形成一层不溶于培养液的保护膜。
(3)成形
将含脂肪干细胞、胰岛素、地塞米松、抑肽酶和异丁基甲基化黄嘌呤衍生物的海藻酸钠‐明胶水凝胶和不含细胞的水凝胶分别装入第一挤压式喷头113和第二挤压式喷头114，内皮细胞‐脂肪干细胞悬液和神经细胞‐脂肪干细胞悬液分别装入第一喷洒式喷头104和第二喷洒式喷头105，氯化钙交联剂装入第四喷洒式喷头115，合成高分子装入第三喷洒式喷头108。将带明胶内芯的支架安装在成形容器102内并与电机输出轴相连，并在成形容器内装入100mL的细胞培养液。整个装置置于温度为10～12℃的环境中，导入乳房模型后启动成形装置，开始成形。
(4)溶芯
成形完毕后，将含明胶薄膜内芯的乳房前体置于恒温箱中培养，使明胶内芯“融化”，取出支架，即得到乳房前体。
(5)后期培养
培养过程中，细胞间建立连接并增殖生长，在不含细胞的凝胶丝周围的内皮(神经)细胞和脂肪干细胞诱导分化形成内径约为1mm的血管(神经)，在凝胶降解后，最终形成具有一定功能的乳房。

Claims (7)

  1. 利用旋转堆积法制备组织器官的专用设备，包括支撑装置、多喷头组件、运动机构、成形装置以及控制系统；所述支撑装置包括支撑腿和底板(101)；所述运动机构采用导轨‐滑块机构，包括X向导轨(121)、X向滑块(120)、Y向导轨(107)、第一Y向滑块(111)、第二Y向滑块(112)、Z向导轨(118)、Z向滑块(106)；所述X向导轨(121)设在底板(101)上，Z向导轨(118)固接在X向滑块(120)上，Y向导轨(107)固接在Z向滑块(106)上；其特征在于：所述多喷头组件至少含有六个喷头，分两组分别安装在第一Y向滑块和第二Y向滑块上；所述成形装置包括成形容器(102)、支架(103)和电机(119)，成形容器(102)安装在底板(101)上并处于喷头组件的下方，支架(103)安装在成形容器(102)内并与电机(119)输出轴相连。
  2. 如权利要求1所述的利用旋转堆积法制备组织器官的专用设备，其特征在于：所述支架为两端呈圆柱状、中间为棱柱状结构。
  3. 如权利要求1所述的利用旋转堆积法制备组织器官的专用设备，其特征在于，所述支架的材料为不锈钢或者钛合金。
  4. 如权利要求1所述的利用旋转堆积法制备组织器官的专用设备，其特征在于：所述第一Y向滑块(111)和第二Y向滑块(112)上分别开有与Y向导轨平行的第一小导轨(110)和第二小导轨(117)，第一小导轨和第二小导轨上分别安装第一小滑块(109)和第二小滑块(116)，第一小滑块和第二小滑块上开有固定喷头的孔。
  5. 如权利要求1所述的利用旋转堆积法制备组织器官的专用设备，其特征在于：所述多喷头组件包括至少两个挤压式喷头和四个喷洒式喷头，两个挤压式喷头的喷嘴内径分别为200μm～1mm和50μm～500μm，喷洒式喷头喷嘴的网孔直径为50μm～100μm。
  6. 一种利用旋转堆积法制备组织器官的方法，其特征在于该方法包括以下步骤：
    1)材料制备：
    制备0.2～20％(w/v)水凝胶溶液、1～10％(w/v)交联剂/聚合液、0.1～10％(w/v)合成高分子溶液、0.2～10％(w/v)明胶溶液和细胞培养液；分离或者诱导种子细胞，种子细胞密度为1×103～1×106个/mL，将种子细胞与水凝胶溶液按1～9：9～1体积比混合制成含种子细胞的水凝胶溶液；分离或者诱导内皮细胞和神经细胞并制成细胞悬液，内皮细胞悬液和神经细胞悬液密度均为1×105～1×108个/mL；
    2)内芯制备
    当制备具有内腔的组织器官时，根据所要制备组织器官的内腔形状，用三维绘图软件设计其三维模型，用快速成形法制作具有此内腔结构的模具，当成形实体的组织器官时，模具是直径厚8～10mm的圆柱；将支架(103)放入模具中央，往模具中加入明胶溶液后降低温度至8～10℃使明胶固化，打开模具得到中心带支架(103)的组织器官内芯(401)，在带支架的组织器官内芯(401)外涂覆一层水凝胶溶液，并用交联剂/聚合液交联/聚合；
    3)成形
    将含种子细胞的水凝胶溶液和不含细胞的水凝胶溶液分别装入第一挤压式喷头(113)和第二挤压式喷头(114)中，交联剂/聚合液、神经细胞悬液、内皮细胞悬液以及合成高分子溶液分别装入第四喷洒式喷头(115)、第二喷洒式喷头(105)、第一喷洒式喷头(104)和第三喷洒式喷头(108)中，在成形容器内装入细胞培养液(405)，将支架(103)安装在成形容器(102)内并与电机(119)输出轴相连，将待成形组织器官的模型导入控制系统后，启动成形设备，开始堆积成形；在成形过程中，支架(103)绕Y负向做旋转运动，喷头做平移运动，当成形组织器官的主体部分时，第一挤压式喷头(113)运动到指定位置挤出含种子细胞的水凝胶缠绕在内芯上，同时装有交联剂/聚合液的第四喷洒式喷头(115)运动到第一挤压式喷头(113)的正后方喷洒出交联剂/聚合液对其进行交联/聚合；当成形血管时，第二挤压式喷头(114)运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头(115)运动到第二挤压式喷头(114)正后方，第一喷洒式喷头(104)和第三喷洒式喷头(108)分别运动到第二挤压式喷头(114)正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头(115)下进行交联/聚合，经过培养液进行洗涤，至第一喷洒式喷头(104)、第三喷洒式喷头(108)下进行喷洒内皮细胞和复合合成高分子溶液；当成形神经时，第二挤压式喷头(114)运动到指定位置挤出不含细胞的水凝胶缠绕在内芯上，同时第四喷洒式喷头(115)运动到第二挤压式喷头(114)正后方，第二喷洒式喷头(105)和第三喷洒式喷头(108)分别运动到第二挤压式喷头(114)正前方，在旋转的内芯带动下，新缠绕的水凝胶依次运动至第四喷洒式喷头(115)下进行交联/聚合，经过培养液进行洗涤，至第二喷洒式喷头(105)、第三喷洒式喷头(108)下进行喷洒神经细胞和复合合成高分子；如此反复缠绕堆积直至形成含内芯的组织器官前体；
    4)后续处理
    将含内芯的组织器官前体置于恒温箱中培养，溶除内芯后取出支架，继续培养一段时间后形成回转状组织器官，神经和血管(502)以螺旋状贯穿其中。
  7. 如权利要求6所述的一种利用旋转堆积法制备组织器官的方法，其特征在于：所述的种子细胞为具有分化能力的干细胞或具有生理活性的成体细胞，所述干细胞为脂肪干细胞、胚胎干细胞、血液干细胞、骨髓干细胞或诱导型多功能干细胞，所述成体细胞为脂肪细胞、肝细胞、膀胱细胞、淋巴细胞、心肌细胞或肾细胞；所述水凝胶为复合了明胶、胶原、基质胶、卡拉胶、壳聚糖、琼脂、透明质酸、基质胶、弹性蛋白中层粘素中一种或多种的海藻酸钠或纤维蛋白原溶液；所述的水凝胶中复合各种细胞冻存剂、细胞生长因子、药物、抗凝血剂；细胞冻存剂为二甲基亚砜、甘油、右旋糖中的一种或多种复合物；所述的细胞生长因子为血管内皮生长因子、碱性成纤维细胞生长因子、肝细胞生长因子、人血小板衍化生长因子、转化生长因子β1中的一种或多种复合物；所述的药物为抗肿瘤药物，该抗肿瘤药物为黄芪多糖、顺式二氨基二氯络铂、丝裂霉素、5-氟尿嘧啶、抗过敏药物、抗生素和病毒疫苗中的一种或多种复合物；所述的抗凝血因子为肝素和紫杉醇中的一种或两种复合物；所述合成高分子溶液的溶质为聚氨酯、聚己内酯、聚碳酸酯、聚乙二醇、聚乳酸-羟基乙酸共聚物、聚酯和聚羟基酸酯中的一种或者多种，溶剂为四乙二醇或1,4-二氧六环。
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CN114456929A (zh) * 2021-11-25 2022-05-10 浙江大学 一种三维支架内细胞定域化负载模型的构建装置和方法
CN114789046A (zh) * 2022-05-23 2022-07-26 燕山大学 一种重金属捕集剂及其应用
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