WO2019113901A1 - 一种人造胰岛组织及其制备和应用 - Google Patents

一种人造胰岛组织及其制备和应用 Download PDF

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WO2019113901A1
WO2019113901A1 PCT/CN2017/116250 CN2017116250W WO2019113901A1 WO 2019113901 A1 WO2019113901 A1 WO 2019113901A1 CN 2017116250 W CN2017116250 W CN 2017116250W WO 2019113901 A1 WO2019113901 A1 WO 2019113901A1
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islet
tissue
islet tissue
artificial
biocompatible scaffold
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PCT/CN2017/116250
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English (en)
French (fr)
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张键
薛丽
赵华山
黄晨
杨雅莉
汪宝蓓
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深圳先进技术研究院
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Priority to PCT/CN2017/116250 priority Critical patent/WO2019113901A1/zh
Publication of WO2019113901A1 publication Critical patent/WO2019113901A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells

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  • the present invention relates to the field of biotechnology, and in particular to artificial islet tissue.
  • Islet is the endocrine part of the pancreas. It presents cell clusters of different sizes and ellipsoids in the pancreas.
  • the secreted insulin and glucagon directly participate in the regulation of glucose metabolism in the body.
  • Islet cells are mainly classified into ⁇ cells, ⁇ cells, ⁇ cells, and PP cells. Among them, ⁇ cells account for 60%-70% of islet tissue and secrete insulin. Insulin can act on target organs such as liver, muscle, fat, accelerate the storage and metabolism of glucose, and inhibit the production of glucose. It is the only hypoglycemic hormone in the human body. Insufficient insulin secretion leads to the development of insulin-dependent diabetes mellitus (type I diabetes).
  • the present invention develops an artificial islet tissue based on biological 3D printing technology.
  • the construction of bioactive 3D printed islet tissue not only contributes to in vitro or in vivo diabetes research models, but also provides new therapeutic strategies for insulin-dependent diabetes.
  • the present invention prints biologically functional 3D islet tissue in vitro on the basis of islet cell culture, combined with tissue engineering method and current advanced 3D printing technology, and inoculates islet cells in liquid scaffold material. Co-culture, or direct mixed printing to form a three-dimensional islet tissue-like structure.
  • An artificial islet tissue produced by biocompatible scaffold material and islet cells by 3D bioprinting.
  • the 3D biological printing method is:
  • Biocompatible scaffold material is made into a biocompatible scaffold by 3D bioprinting, and the mixed culture medium containing islet cells is injected into the biocompatible scaffold;
  • the biocompatible scaffold material is mixed with a mixed culture medium containing islet cells to produce artificial islet tissue by 3D bioprinting.
  • the mixed culture liquid is a fluid liquid for culturing the islet cells
  • the mixed culture solution contains animal serum and an antibiotic, and more preferably, the animal serum is selected from the group consisting of RPMI1640, FBS.
  • the biocompatible scaffold material is selected from one or a combination of gelatin, collagen, sodium alginate, agarose, matrigel, hyaluronic acid, chitosan, and dextran.
  • the collagen is selected from the group consisting of fibroblast collagen, more preferably one of type I collagen, type II collagen, type III collagen, type XI collagen, type XXIV collagen, and type XXVII collagen. Or any combination of several.
  • the artificial islet tissue is made to have voids by 3D bioprinting, and the diameter of the voids is not more than 1.5 mm, more preferably not more than 1 mm.
  • Another aspect of the invention provides a method of making artificial islet tissue comprising the steps of:
  • the biocompatible scaffold material is mixed with the mixed culture medium containing islet cells to prepare artificial islet tissue by 3D bioprinting.
  • the method for preparing the artificial islet tissue may further include the step 5), and the prepared artificial islet tissue is cultured in the mixed culture solution.
  • Another aspect of the invention provides artificial islet tissue for the treatment of type 1 diabetes, type 2 diabetes, hyperglycemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complications, obesity, impaired glucose tolerance, insulin resistance Use of islet hyperfunction, islet tissue physics or chemical damage.
  • Another aspect of the invention provides artificial islet tissue for the treatment of type 1 diabetes, type 2 diabetes, hyperglycemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complications, obesity, impaired glucose tolerance, insulin resistance Use in medical devices with islet hyperfunction, islet tissue physics or chemical damage.
  • Another aspect of the invention provides the use of artificial islet tissue in the preparation of a medical model for islet tissue for research and for a directly related disease or indirectly related disease of islet secreting hormone.
  • Directly related diseases or indirectly related diseases of islet secreting hormone are preferably type I diabetes, type II diabetes, hyperglycemia, postprandial hyperglycemia, impaired glucose tolerance, diabetic complications, obesity, impaired glucose tolerance, insulin resistance, islet Hyperfunction, islet tissue physical or chemical damage.
  • Another aspect of the invention provides the use of artificial islet tissue in the preparation of a medical model for studying islet tissue and islet secreting hormone.
  • the biocompatible scaffold material is selected from one or a combination of gelatin, collagen, sodium alginate, agarose, hyaluronic acid, matrigel, dextran and chitosan.
  • gelatin is used as a scaffold material;
  • the mixed culture liquid is a fluid liquid made of a medium and a biologically active substance required to promote growth of islet cells;
  • Islet tissue mixed culture medium RPMI1640 + 10% FBS + 1% penicillin + 1% streptomycin.
  • Islet function assay medium (KRBH buffer) components 118.5 mM NaCl, 4.74 mM KCl, 25 mM NaHCO 3 , 1.19 mM MgSO 4 , 2.54 mM CaCl 2 , 1.19 mM KH 2 PO 4 , 10 mM HEPES, 0.1% BSA.
  • the islet cells may be single islet cells, or mixed cells containing single islet cells, or islet cell clusters containing multiple islet cells, or islet tissue fragments, islet cells of the present invention.
  • the cells derived from the subject or commercially available cells may also be derived from test animals and the like.
  • the islet cells are selected from the group consisting of mice, which are obtained by collagenase perfusion and density gradient centrifugation. After overnight, the islets with moderate volume, intact structure and clear and smooth edge envelope are further digested until A single cell mixture is formed.
  • sources of islet cells are also commercially available.
  • the present invention provides an artificial islet tissue prepared by biocompatible scaffold material, islet cells, and mixed culture solution by 3D bio-printing;
  • the biocompatible scaffold material is made into a scaffold by 3D bioprinting, and the mixed culture solution containing the islet cells is injected into the scaffold; or
  • the artificial islet tissue is prepared by 3D bioprinting
  • the artificial islet cells are made to have voids by 3D bioprinting, and the diameter of the voids does not exceed 1 mm.
  • the size of the void has one or more sizes, and preferably has two to three sizes.
  • the 3D printing size of the biocompatible stent of the present invention is: pore size (R): 50 ⁇ m to 800 ⁇ m; line stacking angle: 0-90°.
  • the pore size (R) is preferably 100 ⁇ m, 150 ⁇ m, 200 ⁇ m, 250 ⁇ m, 300 ⁇ m, 350 ⁇ m, 400 ⁇ m, 450 ⁇ m, 500 ⁇ m, 550 ⁇ m, 600 ⁇ m, 650 ⁇ m, 700 ⁇ m, 750 ⁇ m;
  • the angle of line stacking is preferably 5°, 10°, 15°, 20°, 30°, 40°, 50°, 60°, 70°, 80°, 90°.
  • the invention provides a method for preparing artificial islet tissue, which comprises the following steps:
  • the biocompatible scaffold material is made into a three-dimensional scaffold by 3D bioprinting, and then the mixture liquid containing the islet tissue cells is injected into the scaffold to make artificial islet tissue; or
  • the prepared islet tissue is cultured and stored in a mixed culture solution for use.
  • the artificial islet tissue of the present invention can be used as a treatment tool for a disease directly related to glucose metabolism or an indirectly related disease caused by any factor;
  • the artificial islet tissue of the present invention can be used as a research model for diabetes.
  • Figure 1 Schematic diagram of artificial islet tissue.
  • FIG. 1 Effect of islet tissue cells on insulin secretion levels at different glucose concentrations.
  • Figure 3 Histological morphology of islet tissue.
  • the composition of the mixed culture solution was: RPMI1640 + 10% FBS + 1% penicillin + 1% streptomycin, and the culture solution was equilibrated in a 37 ° C, 5% CO 2 incubator for 2-4 hours.
  • mice were sacrificed by cervical dislocation, and the entrance end of the common bile duct was ligated.
  • the common bile duct was perfused with collagenase P 2.4 mg/head. After the pancreas was filled, carefully and completely removed. The intact pancreas was placed in pre-cooled 6 ml HBSS buffer.
  • the shape of the three-dimensional islet tissue is designed to resemble the shape of the pancreas, and the internal structure is ensured to communicate with a certain pore structure;
  • bracket material is selected to be printed with gelatin.
  • the prepared stent was stored at -80 ° C for use.
  • Each mouse contains 600-4000 islet tissues in the pancreas, and each islet tissue contains about 1000 islet cells.
  • it is made into a three-dimensional scaffold by 3D bioprinting (for example: 2mm ⁇ 2mm ⁇ 1mm), and then contains islets.
  • tissue containing about 1.5 ⁇ 10 6 islets cells was injected into the mixture to produce an artificial islet tissue within the stent.
  • Example 4 In vitro hormone detection and histological detection of artificial islet tissue
  • the human islet tissue was starved for 1 h with 2.8 mM glucose KRBH buffer, and the artificial pancreatic tissue was treated with 2.8 mM/16.7 mM glucose KRBH buffer for 4 h, and the supernatant was taken.
  • the insulin secretion of artificial pancreatic tissue cultured in vitro was detected by ELISA.
  • Level and glucose stimulate insulin secretion sensitivity. It has been proved by experiments that the islet cells used have the ability to secrete insulin. Compared with low glucose conditions, high glucose stimulation can effectively increase the insulin content in the culture medium and have the ability to regulate blood sugar levels in vivo.
  • Example 5 In vivo transplantation experiment of artificial islet tissue
  • the artificial islet tissue prepared by the invention can be used for a transplantation test, in particular, after the adult type 1 diabetes model mouse is anesthetized, the prepared islet tissue is transplanted into the subcutaneous or renal cyst of the mouse.

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Abstract

一种人造胰岛组织，包括生物相容性支架、胰岛细胞以及混合培养液，通过3D生物打印技术，将生物相容性支架与胰岛组织制成具有生物活性的人造胰岛组织。混合培养液用于胰岛细胞及组织的培养和保存。生物相容性支架以3D打印方式制成。人造胰岛组织可作为糖尿病的病理生理机制研究及治疗工具。

Description

一种人造胰岛组织及其制备和应用 技术领域
本发明涉及生物技术领域，具体涉及人造胰岛组织。
背景技术
胰岛是胰腺的内分泌部，在胰腺中呈现大小不同、椭球形的细胞团簇，其分泌的胰岛素和胰高血糖素直接参与机体糖代谢的调节。胰岛细胞主要分为α细胞、β细胞、γ细胞及PP细胞等。其中β细胞占胰岛组织的60％‐70％，分泌胰岛素。胰岛素能作用于肝脏，肌肉，脂肪等靶器官，加速葡萄糖的储存与代谢，并抑制葡萄糖的生成，是人体内的唯一降糖激素。胰岛素分泌不足导致胰岛素依赖型糖尿病(I型糖尿病)的发生。
目前对于糖尿病尚无根治的方法，对于胰岛素依赖的患者，唯一的办法就是胰岛素药物注射，然而这种方式，不仅涉及每天定时定量的注射，而且，也不可避免药物依赖以及药物的毒副作用，由此可见，开发一种简单一次性具有生物自我调节能力的胰岛组织假体，将是一种有前景的糖尿病治疗手段，因此，本发明基于生物3D打印技术，开发了一种人造胰岛组织，人工构建具有生物活性的3D打印胰岛组织，不仅有助于体外或体内糖尿病研究模型，而且也为胰岛素依赖性糖尿病提供了新的治疗策略。
发明内容
为了解决上述提到的问题，本发明在胰岛细胞培养的基础上，结合组织工程方法和目前先进的3D打印技术，体外打印具有生物学功能的3D胰岛组织，将胰岛细胞接种在液态支架材料进行共培养，或直接混合打印方式形成三维胰岛组织样结构。
一种人造胰岛组织，其由生物相容性支架材料、胰岛细胞通过3D生物打印方式制成。
在本发明的技术方案中，通过3D生物打印方式为：
将生物相容性支架材料通过3D生物打印制成生物相容性支架，再将含胰岛细胞的混合培养液注入生物相容性支架上；或者
生物相容性支架材料与含胰岛细胞的混合培养液进行混合，通过3D生物打 印制成人造胰岛组织。
在本发明的技术方案，混合培养液是用于培养胰岛细胞的流动性液体；
优选地，混合培养液中包含动物血清和抗生素，更优选地，所述的动物血清选自RPMI1640、FBS。
在本发明的技术方案中，生物相容性支架材料选用明胶、胶原蛋白、海藻酸钠、琼脂糖、基质胶、透明质酸、壳聚糖、葡聚糖中的一种或者几种任意组合；优选地，所述胶原蛋白选自成纤维胶原，更优选为I型胶原蛋白、II型胶原蛋白、III型胶原蛋白、XI型胶原蛋白、XXIV型胶原蛋白、XXVII型胶原蛋白中的一种或者几种任意组合。
在本发明的技术方案中，通过3D生物打印制成人造胰岛组织上具有空隙，空隙的直径不超过1.5mm，更优选不超过1mm。
本发明另一个方面提供了人造胰岛组织的制备方法，其包括以下步骤：
1)制备生物相容性支架材料，
2)制备混合培养液；
3)分离胰岛细胞，并以混合培养液分别进行培养；
4)将生物相容性支架材料通过3D生物打印制成生物相容性支架，再将含胰岛细胞的混合培养液注入生物相容性支架上；或者
4)生物相容性支架材料与含胰岛细胞的混合培养液进行混合，通过3D生物打印制成人造胰岛组织。
在本发明的技术方案中，人造胰岛组织的制备方法还可以包括步骤5)，将制备得到的人造胰岛组织在混合培养液中进行培养。
本发明的另一个方面提供了人造胰岛组织在用于治疗I型糖尿病、II型糖尿病、高糖血症、餐后高血糖、葡萄糖耐量障碍、糖尿病并发症、肥胖症、糖耐量减低、胰岛素抵抗、胰岛功能亢进、胰岛组织物理或化学损伤中的用途。
本发明的另一个方面提供了人造胰岛组织在用于治疗I型糖尿病、II型糖尿病、高糖血症、餐后高血糖、葡萄糖耐量障碍、糖尿病并发症、肥胖症、糖耐量减低、胰岛素抵抗、胰岛功能亢进、胰岛组织物理或化学损伤的医疗器械中的用途。
本发明的另一个方面提供了人造胰岛组织在制备用于研究的胰岛组织以及胰岛分泌激素的直接相关疾病或间接相关疾病的医学模型中的用途。
胰岛分泌激素的直接相关疾病或间接相关疾病优选为I型糖尿病、II型糖尿病、高糖血症、餐后高血糖、葡萄糖耐量障碍、糖尿病并发症、肥胖症、糖耐量减低、胰岛素抵抗、胰岛功能亢进、胰岛组织物理或化学损伤。
本发明的另一个方面提供了人造胰岛组织在制备用于研究胰岛组织以及胰岛分泌激素的医学模型中的用途。
具体发明内容如下：
1.支架材料选择
在本发明的技术方案中，生物相容性支架材料选用明胶、胶原蛋白、海藻酸钠、琼脂糖、透明质酸、基质胶、葡聚糖、壳聚糖中的一种或者几种任意组合；优选地，本发明的一个优选的实施方案中以明胶为支架材料；
2.培养基选择
混合培养液是由培养基和促进胰岛细胞生长所需的生物活性物质制成的流动性液体；
胰岛组织混合培养液：RPMI1640+10％FBS+1％青霉素+1％链霉素。胰岛功能检测培养液(KRBH缓冲液)成分：118.5mM NaCl、4.74mM KCl、25mM NaHCO 3、1.19mM MgSO 4、2.54mM CaCl 2、1.19mM KH 2PO 4、10mM HEPES、0.1％BSA。
3.胰岛细胞选择
在本发明的技术方案中，所述胰岛细胞可以为单一胰岛细胞，或者包含单一胰岛细胞的混合细胞，或者包含多个胰岛细胞的胰岛细胞簇，或者胰岛组织碎片，本发明所述的胰岛细胞为受试者来源的细胞或者市售细胞，也可以来源于试验动物等。
在本发明的一个技术方案中，所述胰岛细胞选自小鼠，经胶原酶灌注及密度梯度离心法分离获得，过夜后选择体积适中，结构完整，边缘包膜清晰光滑的胰岛进行进一步消化直至形成单细胞混合液。除此之外，胰岛细胞的来源也可以通过市售获得。
4.打印方式
本发明提供了一种人造胰岛组织，其由生物相容性支架材料、胰岛细胞、以及混合培养液通过3D生物打印方式制成；
在本发明的方案中，将生物相容性支架材料通过3D生物打印制成支架，再 将含胰岛细胞的混合培养液注入支架上；或者
生物相容性支架材料与胰岛细胞混合培养液进行混合后，通过3D生物打印制成人造胰岛组织；
5.打印参数选择
在本发明的技术方案中，通过3D生物打印制成人造胰岛细胞上具有空隙，空隙的直径不超过1mm。
在本发明的技术方案中，所述空隙的尺寸具有1种以上的尺寸，优选具有2-3种尺寸。
在一个优选的技术方案中，本发明生物相容性支架的3D打印尺寸为：孔隙尺寸(R)：50μm～800μm；线条堆积夹角:0-90°。
孔隙尺寸(R)优选为100μm、150μm、200μm、250μm、300μm、350μm、400μm、450μm、500μm、550μm、600μm、650μm、700μm、750μm；
线条堆积夹角优选为5°、10°、15°、20°、30°、40°、50°、60°、70°、80°、90°。
6.3D打印人造胰岛组织的制备过程
本发明提供了人造胰岛组织的制备方法，其包括以下步骤：
1)制备生物相容性支架材料；
2)制备混合培养液；
3)分离胰岛细胞，并以混合培养液分别进行培养；
4)先将生物相容性支架材料，通过3D生物打印制成三维支架，再将含胰岛组织细胞的混合物液注入支架内制成人造胰岛组织；或者
4)生物相容性支架材料与胰岛组织细胞混合物混合，通过3D生物打印制成人造胰岛组织；
5)将制备得到的胰岛组织在混合培养液中进行培养保存备用。
7.人造胰岛组织应用
1)体外激素检测：用ELISA方法，检测体外培养的人造胰岛组织的胰岛素分泌水平；或者
2)体内移植实验：将制备好的胰岛组织移植于糖尿病模型小鼠中，随后检测小鼠血糖水平及胰岛素分泌水平。
有益效果
1)本发明的人造胰岛组织可作为任何因素引起的糖代谢直接相关疾病或间接相关疾病的治疗工具；
2)本发明的人造胰岛组织可作为糖尿病的研究模型。
附图说明
图1：人造胰岛组织示意图。
图2：不同葡萄糖浓度下胰岛组织细胞对胰岛素分泌水平的影响。
图3：胰岛组织组织学形态。
具体实施方式
下列实施例描述了本发明的具体方面以便解释本发明并且为本领域技术人员提供有关本发明方法的描述。这些实施例不应用来限定本发明，因为这些实施例仅提供了用于理解和实施本发明及其各个方面的具体方法。
实施例1：混合培养液的配置
混合培养液的成分为：RPMI1640+10％FBS+1％青霉素+1％链霉素，培养液放入37℃，5％CO 2的培养箱内平衡2-4小时。
实施例2：胰岛细胞获得
断颈处死小鼠，结扎胆总管十二指肠入口端，胆总管灌注胶原酶P 2.4mg/只，待胰腺充盈后小心完整取出；将取出的完整胰腺放入预冷6ml HBSS缓冲液中，37℃震荡消化12min；消化结束后加入预冷10％FBS RPMI1640培养基终止消化，大力摇晃15s后4℃800rpm离心4min去上清；10％FBS RPMI1640培养基重悬沉淀后50目金属筛网过滤，滤液4℃800rpm离心4min去上清；10ml histopaque1077重悬沉淀并缓慢加入10ml无血清RPMI1640培养基使两溶液存在清晰分层界面；20℃2400rpm离心20分钟，吸取两层溶液界面之间组织细胞团，10％FBS RPMI 1640培养基洗1-2次，800rpm离心2min；2ml10％FBS RPMI 1640培养基重悬沉淀，75um细胞筛过滤得到胰岛，将胰岛移入6cm培养皿中，RPMI1640+10％FBS+1％青霉素+1％链霉素培养过夜。过夜后选择体积适中，结构完整，边缘包膜清晰光滑的胰岛作为3D打印原材料。并在1天内完成3D人造胰岛组织的制备(如图1所示)。
实施例3：3D打印胰岛组织的制备
1.生物支架的设计及制备
(1)结合计算机辅助设计(Computer aided design,CAD)，采用三维打印技术构建支架，其中形状、组成成分及内部结构都具有良好可设计性；
(2)三维胰岛组织的形状设计为类似胰腺的形状，并保证内部结构联通，具有一定孔隙结构；
(3)支架材料选择明胶配伍打印。
(4)所构建三维胰岛组织内部结构参数：孔隙尺寸(R)：＝0.2mm；线条堆积夹角：90°。
(5)将制备好的支架置于-80℃储存备用。
2.制备3D人造胰岛组织
每只小鼠胰腺中含有600-4000个胰岛组织，每个胰岛组织中含有1000左右的胰岛细胞，打印时通过3D生物打印制成三维支架(如：2mm×2mm×1mm)，再将含胰岛组织细胞(约含1.5×10 6个胰岛细胞)的混合物液注入支架内制成人造胰岛组织。
并短期放入RPMI1640+10％FBS+1％青霉素+1％链霉素培养液中备用。
实施例4：人造胰岛组织体外激素检测和组织学检测
用含2.8mM葡萄糖KRBH缓冲液饥饿人造胰岛组织1h后，分别利用含2.8mM/16.7mM葡萄糖KRBH缓冲液处理人造胰岛组织4h，取上清，通过利用ELISA法检测体外培养的人造胰岛组织胰岛素分泌水平及葡萄糖刺激胰岛素分泌敏感性。经实验证明，所用的胰岛细胞具有分泌胰岛素能力，与低糖条件相比，高糖的刺激能够有效提高培养液中胰岛素的含量，具备体内调节血糖水平的能力，具体结果参见图2，其中，16.7mM葡萄糖组胰岛素含量(3.2ng/ml)较2.8mM葡萄糖组(2.3ng/ml)显著提高。。通过组织切片观察胰岛组织细胞形态，具体试验结果如图3所示。
实施例5：人造胰岛组织体内移植实验
本发明制备的人造胰岛组织可以用于移植试验，具体为将成年1型糖尿病模型小鼠麻醉后，将制备好的胰岛组织移植于小鼠皮下或肾包囊处。
因为可以在不脱离本发明范围和实质的情况下对上述主题进行各种改变，所以意图是上述描述中包含的或所附权利要求中定义的所有主题均解释为对本发明的描述和说明。能够根据上述教导对本发明进行许多修改和变型。

Claims (10)

  1. 一种3D打印人造胰岛组织，其由生物相容性支架材料、胰岛细胞通过3D生物打印方式制成。
  2. 根据权利要求1所述的3D打印人造胰岛组织，通过以下方法获得：
    将生物相容性支架材料通过3D生物打印制成生物相容性支架，再将含胰岛细胞的混合培养液注入生物相容性支架上；或者
    生物相容性支架材料与含胰岛细胞的混合培养液进行混合，通过3D生物打印制成人造胰岛组织。
  3. 根据权利要求1或2所述的3D打印人造胰岛组织的制备方法，其包括以下步骤：
    1)制备生物相容性支架材料，
    2)制备混合培养液；
    3)分离胰岛细胞，并以混合培养液分别进行培养；
    4)将生物相容性支架材料通过3D生物打印制成生物相容性支架，再将含胰岛细胞的混合培养液注入生物相容性支架上；或者
    4)生物相容性支架材料与含胰岛细胞的混合培养液进行混合，通过3D生物打印制成人造胰岛组织。
  4. 根据权利要求3所述的制备方法，还可以包括步骤5)，将制备得到的人造胰岛组织在混合培养液中进行培养。
  5. 根据权利要求1或2所述的3D打印人造胰岛组织或者根据权利要求3-4所述的制备方法，生物相容性支架的3D打印尺寸为：孔隙尺寸(R)：50μm～800μm；线条堆积夹角:0-90°。
  6. 根据权利要求1或2所述的3D打印人造胰岛组织或者根据权利要求3-4所述的制备方法，生物相容性支架材料选用胶原蛋白、海藻酸钠、明胶、琼脂糖、基质胶、透明质酸、壳聚糖、葡聚糖中的一种或者几种任意组合；
    优选地，所述胶原蛋白选自成纤维胶原，更优选为I型胶原蛋白、II型胶原蛋白、III型胶原蛋白、XI型胶原蛋白、XXIV型胶原蛋白、XXVII型胶原蛋白中的一种或者几种任意组合。
  7. 根据权利要求1或2所述的3D打印人造胰岛组织或者根据权利要求3-4所述的制备方法，其中，为单一胰岛细胞，或者包含单一胰岛细胞的混合细胞，或者包含多个胰岛细胞的胰岛细胞簇，或者胰岛组织碎片，优选地，所述胰岛细胞来源于受试者。
  8. 根据权利要求1-2，4-7任一项所述的3D打印人造胰岛组织在用于治疗I型糖尿病、II型糖尿病、高糖血症、餐后高血糖、葡萄糖耐量障碍、糖尿病并发症、肥胖症、糖耐量减低、胰岛素抵抗、胰岛功能亢进、胰岛组织物理或化学损伤中的用途。
  9. 根据权利要求1-2，4-7任一项所述的3D打印人造胰岛组织在用于治疗I型糖尿病、II型糖尿病、高糖血症、餐后高血糖、葡萄糖耐量障碍、糖尿病并发症、肥胖症、糖耐量减低、胰岛素抵抗、胰岛功能亢进、胰岛组织物理或化学损伤的医疗器械中的用途。
  10. 根据权利要求1-2，4-7任一项所述的3D打印人造胰岛组织在制备用于研究的胰岛组织以及胰岛分泌激素的直接相关疾病或间接相关疾病的医学模型中的用途。
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