CN103756955B - The bionical composite construction of a kind of personalization and preparation thereof and for the method for drug screening - Google Patents
The bionical composite construction of a kind of personalization and preparation thereof and for the method for drug screening Download PDFInfo
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- CN103756955B CN103756955B CN201410030653.4A CN201410030653A CN103756955B CN 103756955 B CN103756955 B CN 103756955B CN 201410030653 A CN201410030653 A CN 201410030653A CN 103756955 B CN103756955 B CN 103756955B
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Abstract
The bionical composite construction of personalization and preparation thereof and for the method for drug screening, relate to a kind of bionical composite construction and preparation thereof based on compound many shower nozzles three-dimensional printing technology. This bionical composite construction comprises macromolecule shell, branch vessel support, district of functional organization, district of vascular tissue and polymeric isolation layer. District of functional organization is for containing histiocytic hydrogel layer; District of vascular tissue is the hydrogel layer containing blood vessel seed cell; Arrange at space-alternating in district of functional organization and district of vascular tissue; Polymeric isolation layer is intervally arranged in district of functional organization and district of vascular tissue, and district of functional organization and vascular tissue are distinguished into multiple zonules; Branch vessel support comprises arteries and vein blood vessel two parts. The present invention prepares the histoorgan biomimetic features with branch vessel by compound many shower nozzles 3D printing technique; Hydrogel structure needn't be cross-linked or polymerization; Simulate to greatest extent the state of Liao Tinei blood vessel tissue and blood vessel, for the organ-tissue of vascularization provides reference.
Description
Technical field
The invention belongs to bioengineering field, specific to biology or enzyme, its compositions field, relate to a kind of personalized bionicalComposite construction and preparation thereof and for the method for drug screening.
Background technology
In recent years, along with interdisciplinary interchange contact is day by day tight, cell arrangement technology is arisen at the historic moment, for organizational project,Engineering foundation has been established in organ manufacture and drug screening. Wherein outstanding three-dimensional printing technology, is also Quick-forming (RapidPrototyping, RP) technology, utilize the principle rapidoprint [Fedorovich of Computer Aided Modeling and discrete-pile upNE, etal.Trendsinbiotechnology, 2011; 29 (12): 601-606]. This technology can be accurate by biomaterialLocation, is paid much attention in the assembled in vitro field of complex organ. Tissue or the organ of external shaping are of many uses, can be used forResearch maintains Ji Li [GriffithLG, the etal.NatureReviewsMolecularCell of organ normal morphologyBiology, 2006; 7 (3): 211-224], cancer cell evade the treatment measures such as chemotherapy [YamadaKM, etal.Cell,2007; 130 (4): 601-610], transplanting [GriffithLG, etal.ScienceSignaling, 2002 of diseased organ;295 (5557): 1009] and drug screening. External many scientific research groups have completed the transformation of multiple RP technology, to realize based on numberAssembling or the printing containing cell three-dimensional structure of control. (the Centerof of organ manufacturing center of department of mechanical engineering of domestic Tsing-Hua UniversityOrganManufacturing) develop a series of rapid forming equipments, and utilizing these equipment successfully to prepare letter[WangXH, etal.TrendsinBiotechnology, 2007 such as single rete vasculosum, liver organization and bone renovating material;25:505;WangXH,etal.TissueEngineeringPartB,2010;16:189;WangXH.ArtificialOrgans, 2012; 36:591]. Cell/host material three-dimensional structure after assembling generally needs crosslinked or polymerization natural polymerMaterial keeps the stability of three-dimensional structure.
Since the nineties in last century, the exploitation of new drug mainly detects by the biological information based on molecule and cellular levelPlatform completes, the single cell of planting that mainly utilizes external planar to cultivate. But two dimensional surface is cultivated can not simulate human body completelyThe real-life three-dimensional environment of corresponding many cells (at least three kinds) tissue. Adopting two-dimentional cultured cell is that target cell screensThe medicine overwhelming majority does not have activity [KangXH, etal.TissueEngineering, 2005 in animal body; 11:456-458]. In recent years along with the minute yardstick of biological study, the indices that biologists are increasing finds cells survival asOxygen concentration, existence temperature all have vital impact to the growth conditions of cell, and this also makes the external of tissueSimulation and cultivation are especially challenging. The development of combinatorial chemistry, genomics makes can be used for compound or the medicine of screeningKind sharp increase, the development that forces the flux of drug screening to grow with each passing hour, confirmatory method in the past becomes no longer suitableWith, high-flux medicaments sifting (HighthroughputScreening, HTS) is therefore suggested and promotes rapidly[RodriguesAD.Pharmaceuticalresearch, 1997; 4 (11): 1504-1510]. But HTS technology adopts single kindThe two dimension of cell is cultivated, and structure function of cell is imperfect, and the follow-up expection pharmacology of tool is not alive for the definite lead compound of this technologyProperty. Adopt a kind of new high medicaments sifting model of the success rate that more meets human structurology---High content screening (HighContentScreening, HCS) technology determines that lead compound becomes the common recognition in drug research field. Cell need to be integratedFrom the information that provides of the various materials such as DNA, RNA, protein, metabolite and ion, and show individual features; And ion, generationThanking product, large molecule and the interaction of organelle on time and space is the basis that organism is realized complicated vital functions.Therefore, want to understand more clearly the target spot of picking out institute's role in cell function, just one of needs can be certainlyThe instrument of the information that moving extraction target spot expresses in cell, and this instrument is HCS technology [GiulianoKA, etAl.Assayanddrugdevelopmenttechnologies, 2003; 1 (4): 565-577]. With respect to HTS technology,HCS technology can realize many target spots and select. HCS uses fluorescently-labeled antibody, DNA probe, bio-ligand or bioreactorCollect target spot in the intracellular impact on links such as cellular morphology variation, growth, differentiation, apoptosis etc. means, can be oneIn inferior test, obtain the bulk information about each component such as cell DNA, protein. With respect to the HCS skill taking orifice plate as unitArt, HTS the selection result is more accurate, has more biological significance.
Patent (application number 200910079726.8) relates to a kind of Vascularized fat depot and structure side thereof based on subregionMethod, is wrapped in fat granule in micro-capsule, obtains having the three-dimensional adipose tissue structure of communicating structure, and structure is mainly fatDistrict and angiosomes. But the method needs a large amount of crosslinking agents (as CaCl2), can damage cytoactive; The drug main of the methodDischarge by micro-capsule, drug effect is difficult for being applied to total body. Patent (application number 200910155794.8) relates to a kind of sugarThe High content screening method of urinating disease medicine, obtains celliferous three-dimensional structure by RP technique, and to containing pancreasThe three-dimensional structure of island cell is carried out drug screening. But the method is not containing vessel branch system, and vascular endothelial cell is only distributed inHydrogel surface. The method also needs a large amount of crosslinking agents to be stable into body.
Existing organizational project cell/support complex structure body can be expressed tissue or organ in certain dimension in vitroCharacteristic, but can not simulate the cellulous real structure containing vascular system completely. Hydrogel material used in the present inventionCan be cross-linked, because polymeric isolation layer and shell support for structure provides mechanics; Separation layer and shell can beGrand (micro-) sees loose structure, ensured the exchange of nutriment; The three-dimensional very big degree of blood vessel tissue has been simulated vivo environment;Branch vessel structure can be connected with the inside and outside circulatory system, is convenient to HCS drug screening. It is compound many that these factors impel us to utilizeShower nozzle three-dimensional printing technology preparation is simulated the state of intracorporeal organ containing the bionical composite construction of personalization of branch vessel and by itFor drug screening.
Summary of the invention
The object of the invention is the bionical composite construction of a kind of personalization and preparation thereof and the method for drug screening, make itArteries and vein blood vessel state in better analogue body, structure does not need to be cross-linked, and the cell density in structure can be veryHeight, the present invention can be used as bionical composite construction and comes the state of blood vessel tissue in analogue body, and then for drug screening.
A kind of personalized bionical composite construction, is characterized in that: the bionical composite construction of described personalization comprises outside macromoleculeShell, branch vessel support, district of functional organization, district of vascular tissue and polymeric isolation layer; Described district of functional organization is for containing tissueThe hydrogel layer of cell; Described district of vascular tissue is the hydrogel layer containing blood vessel seed cell; District of described functional organization and bloodArrange at space-alternating in tubing district; Described polymeric isolation layer is intervally arranged in district of functional organization and district of vascular tissue,And district of functional organization and vascular tissue are distinguished into multiple zonules; Described branch vessel support comprises arteries and venous bloodPipe two parts, arteries is containing at least one entrance and several branches, and vein blood vessel is containing at least one outlet and several points; Described branch vessel support adopts synthesized polymer material, and branch vessel support is through macromolecule shell, functional organizationBetween district, district of vascular tissue and polymeric isolation layer; Described macromolecule shell adopts natural polymer or synthetic macromolecule materialMaterial.
In technique scheme, the internal diameter of described branch vessel support is 10 μ m～10mm, and wall thickness is 100 μ m～2mm; InstituteThe bed thickness of stating polymeric isolation layer and macromolecule shell is 0.1 μ m～2mm.
Careful containing blood vessel kind containing histiocytic hydrogel layer and district of vascular tissue in district of functional organization of the present inventionBorn of the same parents' hydrogel layer is natural polymer hydrogel, and this natural polymer is gelatin, sodium alginate, fibrinogen, collagen, baseAt least one in matter glue, carragheen, shitosan, agar, hyaluronic acid, matrigel, elastin laminin and laminin; Described naturalIn macromolecule hydrogel, be compounded with at least one in Porcine HGF, heparin, leptin, taxol and Radix Astragali anti-cancer powder.
The mass body volume concentrations of natural polymer hydrogel of the present invention is 0.1～40%; Described blood vessel seed cell andHistiocytic concentration is 1 × 103～1×108Individual/mL; The histocyte in district of described functional organization is adult tissue's cell, one-tenthOne in soma cell and cancer cell; The blood vessel seed cell in district of described vascular tissue is that vascular endothelial cell, fat are dry thinIn born of the same parents, mesenchymal stem cells MSCs, navel blood stem cell, stem cell, embryonic stem cell and induced multi-potent stem cells at leastA kind of.
The preparation method of the bionical composite construction of a kind of personalization provided by the invention, is characterized in that the method comprises followingStep:
1) design branch vessel support threedimensional model, divides two groups and imitates respectively arteries and vein blood vessel;
2) by synthesized polymer material, prepare containing histiocytic hydrogel, prepare containing blood vessel seed cellHydrogel and the synthetic Polymer Solution for preparing be loaded in the different shower nozzles of compound many shower nozzles 3 D-printing equipment;
3) prepare branch vessel support: according to step 1) threedimensional model, utilize compound many shower nozzles 3 D-printing equipment squeezeGo out synthetic Polymer Solution, and freeze-drying removal organic solvent, or melt extrude synthesized polymer material, obtain branch vessel and prop upFrame;
4) prepare district of functional organization: utilize compound many shower nozzles 3 D-printing equipment successively to print to prepare containing histocyteHydrogel, printing path is along branch vessel support, print at interval, obtains preliminary three-dimensional structure;
5) prepare district of vascular tissue: utilize compound many shower nozzles 3 D-printing equipment successively to print, by the blood vessel that contains preparingThe hydrogel of seed cell is filled in the space of preliminary three-dimensional structure, three-dimensional structure in the middle of obtaining;
6) prepare polymeric isolation layer and macromolecule shell: in step 4) and step 5) successively in print procedure, printingAfter one deck or some layers, utilize the synthetic Polymer Solution of compound many shower nozzles 3 D-printing equipment spraying and extract organic solvent, or moltenMelt and extrude synthesized polymer material, form polymeric isolation layer, obtain the friendship of celliferous hydrogel layer and polymeric isolation layerFor structure; Synthesize Polymer Solution and extract organic solvent in the peripheral spraying of alternating structure body, or melt extruding synthetic high scoreSub-material, forms macromolecule shell, obtains personalized bionical composite construction precursor;
7) provide growth factor to the bionical composite construction precursor of above-mentioned personalization, realize the vascularization in district of vascular tissue, makeFunctional organization's district's formative tissue, and make the cell in district of vascular tissue and district of functional organization produce contact, finally obtains describedThe bionical composite construction of propertyization.
In preparation method of the present invention, described compound many shower nozzles 3 D-printing equipment adopts and melt extrudes forming techniqueOr low temperature depositing forming technique is prepared branch vessel support, polymeric isolation layer and macromolecule shell; When employing melt extrudesWhen forming technique is prepared branch vessel support, polymeric isolation layer and macromolecule shell, described synthesized polymer material is thirdAt least one in alkene nitrile-butadiene-styrene, polyurethane, polytetrafluoroethylene (PTFE) and carbohydrate; When adopting low temperature depositing forming techniqueWhile preparing branch vessel support, polymeric isolation layer and macromolecule shell, the solute of described synthetic Polymer Solution is poly-ammoniaEster, Merlon, polyureas ester, PGA, poly-succinic ester, polyether ester, polyethylene glycol, PLA, polycaprolactone, poly-breastAt least one in acid-co-glycolic acid, polyester and polyhydroxy acid ester, the solvent of described synthetic Polymer Solution is tetremGlycol or Isosorbide-5-Nitrae-dioxane. The mass body volume concentrations of described synthetic Polymer Solution is 1%～30%; Described synthetic macromolecule is moltenIn liquid, be compounded with anticoagulin, this anticoagulin is heparin or taxol.
The bionical composite construction of a kind of personalization provided by the invention, for the method for drug screening, is characterized in that the methodComprise the steps:
1) if the histocyte in district of functional organization adopts adult tissue's cell or adult stem cell, bionical multiple to personalizationClose structure and supply with the nutrient solution containing pathogenic medicine, make the bionical composite construction of above-mentioned personalization produce pathological symptom; If function groupThe histocyte of knitting district adopts cancer cell, does not do pathogenic processing;
2) the bionical composite construction of personalization is supplied with to the nutrient solution containing variety classes and various dose curative drug, in real timeOr follow-up collection nutrient solution;
3) analyze the above-mentioned nutrient solution of having collected, the kind of obtaining medical treatment property medicine, dosage are to described personalized composite constructionEffect, by detect, contrast biological indicator, judge the effect of medicine.
In the method for drug screening of the present invention, described biological indicator is carbohydrate lipid metabolism index, liver functionAt least one in index, renal function index, cancer cell index and vascularization index. Described pathological symptom is diabetes, organInflammation, organ failure or tumour.
The present invention compared with prior art, has the technique effect of following advantage and high-lighting: 1. macromolecule of the present invention everyHydrogel layer is divided into some communities by absciss layer, forms the structure that hydrogel layer and polymeric isolation layer are alternately arranged; Macromolecule everyAbsciss layer supports and biochemical protective effect for hydrogel structure provides mechanics, can ensure the shaping of low concentration of water gel; HydrogelNeedn't be cross-linked, cell density can be enough large. 2. the formation cell communication in district of vascular tissue of the present invention and district of functional organization, intervalAlternately arrange, the state of simulation Liao Tinei blood vessel tissue and blood vessel, for the organ-tissue of vascularization provides reference. 3. thisBright macromolecule shell supports and biochemical protective effect for internal structure provides mechanics, favourable and described personalized biomimetic featuresVivo and vitro is cultivated. 4. branch vessel support of the present invention outlet and entrance can with blood vascular system in external pulsation culture systems or bodySystem connects. 5. the method for drug screening of the present invention can be analyzed the effect of medicine to the bionical composite construction of personalization in time, is beneficial toTo the drug screening work of metabolic disease, organ disease or cancer.
Brief description of the drawings
Fig. 1 is the structural representation of personalized bionical composite construction.
Fig. 2 is the schematic diagram of branch vessel support.
Fig. 3 a and Fig. 3 b are respectively the schematic diagram in district of functional organization and the district of vascular tissue of printing.
Fig. 4 a and Fig. 4 b are respectively the alternating structure in polymeric isolation layer and district of functional organization and district of vascular tissue and quick-friedFried view.
In figure: 101 – arterial blood entrances; 102 – branch vessel supports; 103 districts of – functional organization; 104 districts of – vascular tissue;105 – venous blood outlets; 106 – macromolecule shells; 401 – polymeric isolation layers.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is further described.
Fig. 1 is the structural representation of personalized bionical composite construction, and the bionical composite construction of described personalization comprises macromoleculeShell 106, branch vessel support 102, district of functional organization 103, district of vascular tissue 104 and polymeric isolation layer 401; Described meritCan organize district 103 for containing histiocytic hydrogel layer; Described district of vascular tissue 104 is the hydrogel containing blood vessel seed cellLayer; Arrange at space-alternating in district of described functional organization 103 and district of vascular tissue 104; Described polymeric isolation layer 401 interval rowsCloth, in district of functional organization 103 and district of vascular tissue 104, and is divided into multiple little by district of functional organization 103 and district of vascular tissue 104Region; Described branch vessel support 102 comprises arteries and vein blood vessel two parts, arteries containing at least one entrance andSeveral branches, vein blood vessel is containing at least one outlet and several branches; Described branch vessel support 102 adopts synthetic highMolecular material, branch vessel support 102 is through macromolecule shell 106, district of functional organization 103, district of vascular tissue 104 and high scoreBetween sub-separation layer 401; Described macromolecule shell 106 adopts natural polymer or synthesized polymer material.
The internal diameter of described branch vessel support is 10 μ m～10mm, and wall thickness is 100 μ m～2mm; Described polymeric isolation layerAnd the bed thickness of macromolecule shell is 0.1 μ m～2mm. Fig. 2 is branch vessel support schematic diagram, and containing of district of described functional organization organizedThe hydrogel layer containing blood vessel seed cell in the hydrogel layer of cell and district of vascular tissue is natural polymer hydrogel, and this is naturalMacromolecule is gelatin, sodium alginate, fibrinogen, collagen, matrigel, carragheen, shitosan, agar, hyaluronic acid, matrixAt least one in glue, elastin laminin and laminin; In described natural polymer hydrogel, be compounded with Porcine HGF, liverAt least one in element, leptin, taxol and Radix Astragali anti-cancer powder. The mass body volume concentrations of described natural polymer hydrogel is0.1～40%; Described blood vessel seed cell and histiocytic concentration are 1 × 103～1×108Individual/mL; District of described functional organizationHistocyte be the one in adult tissue's cell, adult stem cell and cancer cell; The blood vessel seed in district of described vascular tissueCell is that vascular endothelial cell, fat stem cell, mesenchymal stem cells MSCs, navel blood stem cell, stem cell, embryo are dry thinAt least one in born of the same parents and induced multi-potent stem cells.
The preparation method of the bionical composite construction of a kind of personalization provided by the invention comprises the following steps: 1) design branch bloodPipe holder threedimensional model, divides two groups and imitates respectively arteries and vein blood vessel; 2) by synthesized polymer material, containing of preparingHistiocytic hydrogel, the hydrogel containing blood vessel seed cell preparing and the synthetic Polymer Solution preparing are loaded intoIn the different shower nozzles of compound many shower nozzles 3 D-printing equipment; 3) prepare branch vessel support: according to step 1) threedimensional model, profitExtrude synthetic Polymer Solution with compound many shower nozzles 3 D-printing equipment, and freeze-drying removes organic solvent, or melt extrude syntheticMacromolecular material, obtains branch vessel support; 4) prepare district of functional organization: utilize compound many shower nozzles 3 D-printing equipment successively to beatWhat seal prepared contains histiocytic hydrogel, and printing path is along branch vessel support, and as shown in Figure 3 a, print at interval, obtainsPreliminary three-dimensional structure; 5) prepare district of vascular tissue: utilize compound many shower nozzles 3 D-printing equipment successively to print, as Fig. 3 b instituteShow, the hydrogel containing blood vessel seed cell preparing is filled in the space of preliminary three-dimensional structure, three-dimensional in the middle of obtainingStructure; 6) prepare polymeric isolation layer and macromolecule shell: in step 4) and step 5) successively in print procedure, printing oneAfter layer or some layers, utilize the synthetic Polymer Solution of compound many shower nozzles 3 D-printing equipment spraying and extract organic solvent, or meltingExtrude synthesized polymer material, form polymeric isolation layer, obtain replacing of celliferous hydrogel layer and polymeric isolation layerStructure; Synthesize Polymer Solution and extract organic solvent in the peripheral spraying of alternating structure body, or melt extruding synthetic macromoleculeMaterial, forms macromolecule shell, obtains personalized bionical composite construction precursor; 7) to the bionical composite construction precursor of above-mentioned personalizationGrowth factor is provided, realizes the vascularization in district of vascular tissue, make functional organization's district's formative tissue, and make district of vascular tissue and functionOrganize the cell in district to produce contact, finally obtain the described bionical composite construction of personalization.
Preparation method described in this, described compound many shower nozzles 3 D-printing equipment adopts and melt extrudes forming technique or lowTemperature deposition formation technology is prepared branch vessel support, polymeric isolation layer and macromolecule shell; When employing melt extrudes shapingWhen technology is prepared branch vessel support, polymeric isolation layer and macromolecule shell, described synthesized polymer material be acrylonitrile-At least one in butadiene-styrene, polyurethane, polytetrafluoroethylene (PTFE) and carbohydrate; When adopting the preparation of low temperature depositing forming techniqueWhen branch vessel support, polymeric isolation layer and macromolecule shell, the solute of described synthetic Polymer Solution is polyurethane, poly-Carbonic ester, polyureas ester, PGA, poly-succinic ester, polyether ester, polyethylene glycol, PLA, polycaprolactone, polylactic acid-glycolic baseAt least one in acetate multipolymer, polyester and polyhydroxy acid ester, the solvent of described synthetic Polymer Solution be TEG orIsosorbide-5-Nitrae-dioxane. The mass body volume concentrations of described synthetic Polymer Solution is 1%～30%; Multiple in described synthetic Polymer SolutionClosed anticoagulin, this anticoagulin is heparin or taxol.
The bionical composite construction of a kind of personalization provided by the invention comprises the steps: 1 for the method for drug screening) ifThe histocyte in district of functional organization adopts adult tissue's cell or adult stem cell, the bionical composite construction of personalization is supplied with and is containedThe nutrient solution of pathogenic medicine, makes the bionical composite construction of above-mentioned personalization produce pathological symptom; If the tissue in district of functional organization is thinBorn of the same parents adopt cancer cell, do not do pathogenic processing; 2) the bionical composite construction of personalization is supplied with containing variety classes and various doseThe nutrient solution of curative drug, real-time or follow-up collection nutrient solution; 3) analyze the above-mentioned nutrient solution of having collected, obtaining medical treatment property medicineKind, the effect of dosage to described personalized composite construction, by detect, contrast biological indicator, judge the effect of medicine.
The method of drug screening of the present invention, described biological indicator is that carbohydrate lipid metabolism index, liver function refer toAt least one in mark, renal function index, cancer cell index and vascularization index. Described pathological symptom is diabetes, organ inflammationDisease, organ failure or tumour.
Enumerate several specific embodiments below, further to understand the present invention.
Embodiment 1: a kind of personalized bionical vascularization hepatic tissue of preparation is also applied to hepatitis medicament screening
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and liver cell (Hep), wherein ADSCAfter subculture, prepare cell suspending liquid; By natural polymer powder and (DMEM, dulbecco'smodifiedeagleMedium) nutrient solution mixes, and obtaining mass body volume concentrations is 10% gelatin solution, the sodium alginate that mass body volume concentrations is 2%The fibrinogen solution that solution and mass body volume concentrations are 1%, by above-mentioned gelatin solution, sodium alginate soln and fibrinOriginal solution volume ratio 2:2:1 obtains natural polymer solution for later use after mixing; By centrifugal to ADSC and Hep suspension, respectively with skySo Polymer Solution mixes, and obtaining cell concentration is 1 × 106The natural polymer solution containing ADSC of individual/mL, and cell is denseDegree is 3 × 106The natural polymer solution containing Hep of individual/mL.
2) preparation of branch vessel support: Solidworks Software for Design branch vessel threedimensional model, divides through delamination softwareAfter analysing, by 3D printing technique melt extruded molten state polytetrafluoroethylene (PTFE) (TEFLON), obtain synthetic fractionated polymer branch vessel and prop upFrame.
3) preparation of macromolecule shell and polymeric isolation layer: adopt equally 3D printing technique screw extruding molten stateTEFLON prepares shell and separation layer; Shell 10 μ m thickness, separation layer thickness 0.5 μ m.
4) the final establishment of external bionic model: utilize compound many shower nozzles three-dimensional printing technology will be containing Hep natural polymerAlong branch vessel support printing-forming, 50% space is reserved at interval; Utilize compound many shower nozzles three-dimensional printing technology to make the sky containing ADSCSo macromolecule is shaped along above-mentioned headspace, and formed body inside can reserving hole channel; Utilize pulsation system to supply with angiogenesisThe factor (VEGF) and HGF (HGF), induction ADSC is divided into capillary endothelial cell or smooth muscle cell; WillThe said structure in vitro culture of pulsing, alternately changes containing the DMEM of VEGF, containing DMEM and the general DMEM of HGF, realizes liver thinBorn of the same parents' organized stable, the development of vascularization and the contact between the two.
5) bringing out and drug screening of hepatitis: utilize pulsation system to supply with containing pathogenic medicine (as bisatin, methylDOPA, furantoin etc.) DMEM induce drug hepatitis, destroy liver cell, detection record biological indicator (as albumin,The amino invertase of G-6-Pase and tyrosine and vascularization expression); Utilize pulsation system to supply with containing therapeutic medicineThe DMEM of thing (as vitamin, reduced glutathione, glycyrrhizic acid preparation or medicine newly developed) treats, record analysisAbove-mentioned biological indicator, obtains drug screening result.
Embodiment 2: a kind of personalized bionical vascularization hepatic tissue of preparation is also applied to hepatitis medicament screening
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and embryonic stem cell (ESC), cultivateAfter going down to posterity, prepare cell suspending liquid; Natural polymer powder is mixed with DMEM nutrient solution, and obtaining mass body volume concentrations is 20%Gelatin solution, sodium alginate soln that mass body volume concentrations is 4% and the mass body volume concentrations fibrinogen solution that is 2%,More than state that after sol solution, sodium alginate soln and fibrinogen solution volume ratio 1:2:1 mix, to obtain natural polymer molten clearlyLiquid is stand-by; ADSC and ESC suspension is centrifugal respectively, mix with natural polymer solution respectively, obtain cell concentration and be 3 ×106The natural polymer solution containing ADSC of individual/mL, and cell concentration is 1 × 105The natural polymer containing ESC of individual/mLSolution.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) prepare natural polymer collagen branch vessel and prop upFrame.
3) step 3 of the preparation of macromolecule shell and polymeric isolation layer: embodiment 1) prepare outside natural polymer collagenShell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing ESCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel, the CaCl that is 5% with mass body volume concentrations by formed body2Solution andThe crosslinked 2min of thrombin solution of 100U/mL; Utilize pulsation system to supply with stage by stage derivant and make ESC be converted into liver cell, lureLead agent containing aFGF, HGF, OSM, dexamethasone, transferrins etc.; Utilize pulsation system to supply with the angiogenesis factor(VEGF) and HGF (HGF), induction ADSC is divided into capillary endothelial cell or smooth muscle cell; By above-mentionedThe structure in vitro culture of pulsing, alternately changes containing the DMEM of VEGF, containing the DMEM of HGF, containing liver cell derivant stage by stageDMEM and general DMEM, realize hepatocellular organized stable, the development of vascularization and the contact between the two.
5) according to the step 4 of embodiment 1) carry out induction generation and the drug screening of hepatitis.
Embodiment 3: a kind of personalized bionical vascularization hepatic tissue of preparation is also applied to hepatitis medicament screening
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC); To in l cell, leadEntering transcription factor (Oct3/4, Sox2, c-Myc and Klf4) reprogrammed is induced multi-potent stem cells (inducedpluripotentStemcell, iPSC), after subculture, prepare cell suspending liquid; By natural polymer powder and DMEM nutrient solution or(phosphatebufferedsaline) PBS solution mixes, and obtains mass body volume concentrations and be 10% gelatin solution, mass bodyVolume concentrations is the fibrinogen solution that 2% sodium alginate soln and mass body volume concentrations are 1%, with gelatin solution, alginic acidIt is stand-by that sodium solution and fibrinogen solution volume ratio 2:2:1 obtain natural polymer after mixing; ADSC and iPSC are outstanding the most at lastSupernatant liquid is centrifugal respectively, and obtaining cell concentration is 2 × 105The natural polymer solution containing ADSC of individual/mL, and cell concentration is3×106The natural polymer solution containing iPSC of individual/mL.
2) step 2 of the preparation of branch vessel support: embodiment 1) the synthetic fractionated polymer branch vessel support of preparation.
3) preparation of macromolecule shell and polymeric isolation layer: material is that mass body volume concentrations is Isosorbide-5-Nitrae-bis-of 5% PLGAOxygen six encircles solution, utilizes multiinjector spraying nozzle assembly, and this solution spraying, between hydrogel layer, is obtained to hydrogel-PLGAThe alternating structure of layer, is separation layer; This solution spraying, to structure periphery, obtains PLGA shell the most at last.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing iPSCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Formed body is immersed to PBS solution, and Isosorbide-5-Nitrae-dioxane is removed in extractionOrganic solvent; Utilize pulsation system to supply with stage by stage derivant and make iPSC be converted into liver cell, derivant is containing activin A, bFGFAnd HGF; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and HGF (HGF), induction ADSC dividesTurn to capillary endothelial cell or smooth muscle cell; By the said structure in vitro culture of pulsing, alternately change containing VEGF'sDMEM, containing the DMEM of HGF, DMEM and the general DMEM of liver cell derivant stage by stage, realize hepatocellular organized stable,The development of vascularization and the contact between the two.
5) according to the step 4 of embodiment 1) carry out induction generation and the drug screening of hepatitis.
Embodiment 4: a kind of personalized bionical vascularization liver cancer tissue of preparation is also applied to liver-cancer medicine screening
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC), buy HCC (HepG2),After subculture, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, and obtaining mass body volume concentrations is 10%Gelatin solution, sodium alginate soln that mass body volume concentrations is 5% and the mass body volume concentrations fibrinogen solution that is 1%,After mixing with gelatin solution, sodium alginate soln and fibrinogen solution volume ratio 3:2:1, obtain natural polymer stand-by; ?ADSC and HepG2 are centrifugal respectively at last, and sneak into respectively in natural polymer solution, and obtaining cell concentration is 3 × 105Individual/mLContaining the natural polymer solution of ADSC, and cell concentration is 3 × 106The natural polymer solution containing HepG2 of individual/mL.
2) preparation of branch vessel support: Solidworks Software for Design branch vessel threedimensional model, divides through delamination softwareAfter analysing, by 3D printing technique screw extruding molten state ABS, obtain branch vessel support.
3) polymeric isolation layer and macromolecule shell preparation: material is that mass body volume concentrations is that the TEG of 5% PU is moltenLiquid, utilizes multiinjector spraying nozzle assembly, and this solution spraying, between hydrogel layer, is obtained to the alternately knot of hydrogel-PU layerStructure, is protective layer; This solution spraying, to structure periphery, obtains PU shell the most at last.
4) utilize compound many shower nozzles 3D printing technique by the branch vessel road of design in advance, natural polymer edge containing HepG2Footpath or computer model carry out 3 D-printing, and interval reserves 50% branch vessel space and refuses cell assembling; Utilize afterwards compoundMany shower nozzles 3D printing technique will be printed along reserved path inside above-mentioned branch vessel containing the natural polymer of ADSC, and at printable layerInner side reserving hole channel; Formed body is immersed to PBS solution, and TEG organic solvent is removed in extraction; Utilize pulsation system to supply with MEM(minimumessentialmedium) nutrient solution nutrient solution is stablized HCC and is made its systematism; Utilize pulsation system to supply with hairThin angiogenesis factor (VEGF) and HGF (HGF), induction ADSC is divided into capillary endothelial cell or flatSliding myocyte; Said structure is proceeded to the in vitro culture of pulsing, alternately change containing the MEM of VEGF, containing the MEM of HGF and generalMEM, realizes organized stable, the development of vascularization of HCC and the contact between the two.
5) liver-cancer medicine screening: the biological indicator of detection record formed body is (as jaundice level, albumin level, indolesThe amino invertase of the green retention rate of cyanogen, G-6-Pase and tyrosine and vascularization expression); Utilize pulsation system to supplyGive curative drug as vitamin, chemotherapy medicine, Sorafenib or medicine newly developed, record and analyze biology and refer toMark, carries out drug screening.
Embodiment 5: prepare the bionical vascularization pancreatic tissue of a kind of personalization and be applied to the drug screening of diabetes
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and beta Cell of islet (Pan β), preparationCell suspending liquid; Natural polymer powder is mixed with DMEM nutrient solution, obtain mass body volume concentrations and be 10% gelatin solution and treatWith; ADSC and Pan β suspension are centrifugal respectively the most at last, sneak into respectively in natural polymer solution, obtain 3 × 106Individual/mL'sContaining the natural polymer solution of ADSC, and 3 × 106The natural polymer solution containing Pan β of individual/mL.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PU support of preparation.
3) step 3 of the preparation of shell and separation layer: embodiment 4) prepare PU shell and separation layer.
4) utilize compound many shower nozzles 3D printing technique by the branch vessel road of design in advance, natural polymer edge containing Pan βFootpath or computer model carry out 3 D-printing, and interval reserves 50% branch vessel space and refuses cell assembling; Utilize afterwards compoundMany shower nozzles 3D printing technique will be printed along reserved path inside above-mentioned branch vessel containing the natural polymer of ADSC, and at printable layerInner side reserving hole channel; Utilize pulsation system supply nutrient solution to stablize beta Cell of islet and make its systematism; Utilize pulsation system to supply with hairThin angiogenesis factor (VEGF) and HGF (HGF), induction ADSC is divided into capillary endothelial cell or flatSliding myocyte; Said structure is proceeded to the in vitro culture of pulsing, alternately change containing the DMEM of VEGF, containing the DMEM and of HGFAs DMEM, realize organized stable, the development of vascularization of beta Cell of islet and the contact between the two.
5) induction generation and the drug screening of diabetes: utilize pulsation system to supply with Streptozotocin(Streptozotocin, STZ) directly destroys beta Cell of islet and causes diabetes B, and now biological indicator of detection record(as glucose consumption, insulin release, C peptide and vascularization expression); Utilize pulsation system to supply with curative drug as sulphurUreas, biguanides, glinides or or medicine newly developed, record and analyze biological indicator, carry out drug screening.
Embodiment 6: prepare the bionical vascularization pancreatic tissue of a kind of personalization and be applied to the drug screening of diabetes
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and embryonic stem cell (ESC), cultivateAfter going down to posterity, prepare cell suspending liquid; Natural polymer is mixed with DMEM nutrient solution, obtain mass body volume concentrations and be 10% brightThe fibrinogen solution that the sodium alginate soln that sol solution, mass body volume concentrations are 2% and mass body volume concentrations are 1%, with brightIt is stand-by that sol solution, sodium alginate soln and fibrinogen solution volume ratio 2:2:1 obtain natural polymer after mixing; The most at lastADSC and ESC are centrifugal respectively, and sneak into respectively in natural polymer solution, and obtaining cell concentration is 3 × 106Individual/mL containsThe natural polymer solution of ADSC, and cell concentration is 3 × 106The natural polymer solution containing ESC of individual/mL.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PEG branch vessel of preparation props upFrame.
3) step 3 of the preparation of shell and separation layer: embodiment 4) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing ESCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilizing pulsation system to supply with stage by stage derivant makes ESC be converted into pancreasIsland β cell, derivant is containing ActivinA, FGF, CYC, RA, CYC, Extendin-4, IGF and Nicotinamide etc.; UtilizePulsation system is supplied with the angiogenesis factor (VEGF) and HGF (HGF), and induction ADSC is divided into blood capillaryEndothelial cell or smooth muscle cell; Said structure is proceeded to the in vitro culture of pulsing, alternately change containing the DMEM of VEGF, containThe DMEM of HGF, containing DMEM and the general DMEM of beta Cell of islet derivant stage by stage, realize the organized steady of beta Cell of isletThe development of vascularization and the contact between the two calmly.
5) according to the step 4 of embodiment 5) carry out induction generation and the drug screening of diabetes.
Embodiment 7: prepare the bionical vascularization pancreatic tissue of a kind of personalization and be applied to the drug screening of diabetes
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC); To in l cell, leadEntering transcription factor (Oct3/4, Sox2, c-Myc and Klf4) reprogrammed is induced multi-potent stem cells (inducedpluripotentStemcell, iPSC), after subculture, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, obtainMass body volume concentrations is 2% sodium alginate soln, and ADSC and iPSC suspension are centrifugal respectively the most at last, and obtaining cell concentration is 3×106The natural polymer solution containing ADSC of individual/mL, and cell concentration is 3 × 106The natural polymer containing iPSC of individual/mLSub-solution.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PCL branch vessel of preparation props upFrame.
3) step 3 of the preparation of shell and separation layer: embodiment 1) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing iPSCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilizing pulsation system to supply with stage by stage derivant is converted into iPSCBeta Cell of islet, derivant is containing ActivinA, FGF, CYC, RA, CYC, Extendin-4, IGF and Nicotinamide etc.; ProfitSupply with the angiogenesis factor (VEGF) and HGF (HGF) by pulsation system, induction ADSC is divided into capillaryVascular endothelial cell or smooth muscle cell; Said structure is proceeded to the in vitro culture of pulsing, alternately change containing the DMEM of VEGF,Containing the DMEM of HGF, containing DMEM and the general DMEM of beta Cell of islet derivant stage by stage, realize the organized steady of beta Cell of isletThe development of vascularization and the contact between the two calmly.
5) according to the step 4 of embodiment 5) carry out induction generation and the drug screening of diabetes.
Embodiment 8: prepare the bionical vascularization Pancreatic Adenocarcinoma of a kind of personalization and be applied to the drug sieve of cancer of pancreasChoosing
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC), buy pancreatic cancer cell (PANC-1),, after subculture, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, obtain mass body volume concentrations and be1% fibrinogen solution, ADSC and PANC-1 are centrifugal respectively the most at last, and sneak into respectively in natural polymer solution, obtainCell concentration is 3 × 106The natural polymer solution containing ADSC of individual/mL, and cell concentration is 3 × 106Individual/mL containsThe natural polymer solution of PANC-1.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PGA branch vessel of preparation props upFrame.
3) preparation of shell and separation layer: embodiment 4 steps 3) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing PANC-1Son carries out 3 D-printing along branch vessel path or the computer model of design in advance, and interval reserves 50% branch vessel spaceRefuse cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along in above-mentioned branch vesselSide reserved path is printed, and inside printable layer reserving hole channel, utilize pulsation system to supply with MEM nutrient solution and stablize pancreatic cancer cellMake its systematism; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and HGF (HGF), inductionADSC is divided into capillary endothelial cell or smooth muscle cell; Said structure is proceeded to the in vitro culture of pulsing, alternately moreChange containing the MEM of VEGF, containing MEM and the general MEM of HGF, realize pancreatic cancer cell organized stable, vascularization development withAnd contact between the two.
5) cancer of pancreas drug screening: the biological indicator of detection record formed body (as glutamic-pyruvic transaminase, glucose, CEA,CA19-9 and vascularization expression); Utilize pulsation system supply with curative drug as gemcitabine, 5-Fu, for Ji Ao etc. orPerson's medicine newly developed, records and analyzes biological indicator, carries out drug screening.
Embodiment 9: prepare the bionical vascularization renal tissue of a kind of personalization and be applied to the drug screening of ephritis
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and Kidney of Newborn cell (NKC), trainingAfter foster going down to posterity, prepare cell suspending liquid; Natural polymer powder is mixed with DMEM nutrient solution, obtain mass body volume concentrations and be20% gelatin solution, by centrifugal to ADSC and NKC suspension, mixes with natural polymer solution respectively, and obtaining cell concentration is 1×106The natural polymer solution containing ADSC of individual/mL, and cell concentration is 2 × 107The natural polymer containing NKC of/mLSolution. Individual
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PHB branch vessel of preparation props upFrame.
3) preparation of shell and separation layer: embodiment 4 steps 3) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing NKCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilize pulsation system supply nutrient solution to stablize nephrocyte and make its tissueChange; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and HGF (HGF), induction ADSC is divided intoCapillary endothelial cell or smooth muscle cell; Be connected and synthesize macromolecule branch support, said structure is proceeded to arteries and veinsMoving in vitro culture, alternately changes containing the DMEM of VEGF, containing DMEM and the general DMEM of HGF, realizes the organized steady of nephrocyteThe development of vascularization and the contact between the two calmly.
5) induction generation and the drug screening of ephritis: utilize pulsation system to supply with antibiotic or non-steroidal anti-inflammatory drugs etc.Destroy NKC, bring out acute interstitial nephritis, and detection record now biological indicator (as glucose, urea, urine albumen creatinineWith vascularization expression); Utilize pulsation system supply with curative drug as hydrochlorothiazide, frusemide, hormone or newly openThe medicine of sending out, records and analyzes biological indicator, carries out drug screening.
Embodiment 10: prepare the bionical vascularization renal tissue of a kind of personalization and be applied to the drug screening of ephritis
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and embryonic stem cell (ESC), cultivateAfter going down to posterity, prepare cell suspending liquid; Natural polymer powder is mixed with DMEM nutrient solution, and obtaining mass body volume concentrations is 10%Gelatin solution, sodium alginate soln that mass body volume concentrations is 2% and the mass body volume concentrations fibrinogen solution that is 1%,More than state that after sol solution, sodium alginate soln and fibrinogen solution volume ratio 2:2:1 mix, to obtain natural polymer molten clearlyLiquid is stand-by; By centrifugal to ADSC and ESC suspension, mix with natural polymer solution respectively, obtaining cell concentration is 1 × 105Individual/The natural polymer solution containing ADSC of mL, and cell concentration is 1 × 105The natural polymer solution containing ESC of individual/mL.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PU branch vessel support of preparation.
3) preparation of macromolecule shell and polymeric isolation layer: embodiment 4 steps 3) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing ESCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilizing pulsation system to supply with stage by stage derivant makes ESC be converted into kidneyCell, derivant is containing FGF-2 and LIF etc.; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and hepatic cell growthThe factor (HGF), induction ADSC is divided into capillary endothelial cell or smooth muscle cell; Said structure is proceeded to pulsatorOuter cultivation, alternately changes containing the DMEM of VEGF, containing the DMEM of HGF, containing DMEM and the general DMEM of nephrocyte derivant, realizes kidneyOrganized stable, the development of vascularization of cell and the contact between the two.
5) according to the step 4 of embodiment 9) carry out induction generation and the ephritis drug screening of ephritis.
Embodiment 11: prepare the bionical vascularization renal tissue of a kind of personalization and be applied to the drug screening of ephritis
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC); To in l cell, leadEntering transcription factor (Oct3/4, Sox2, c-Myc and Klf4) reprogrammed is induced multi-potent stem cells (inducedpluripotentStemcell, iPSC), after subculture, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, obtainMass body volume concentrations is that 10% gelatin solution, sodium alginate soln and the mass body volume concentrations that mass body volume concentrations is 2% are 1%Fibrinogen solution, after mixing with gelatin solution, sodium alginate soln and fibrinogen solution volume ratio 1:2:2, obtainNatural polymer is stand-by; ADSC and iPSC suspension are centrifugal respectively the most at last, and obtaining cell concentration is 1 × 107Individual/mL containsThe natural polymer solution of ADSC, and cell concentration is 3 × 107The natural polymer solution containing iPSC of individual/mL.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule PLGA branch vessel of preparation props upFrame.
3) step 3 of the preparation of macromolecule shell and polymeric isolation layer: embodiment 1) prepare shell and separation layer.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing iPSCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilizing pulsation system to supply with stage by stage derivant is converted into iPSCNephrocyte, derivant is containing FGF-2 and LIF etc.; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and liver cell lifeThe long factor (HGF), induction ADSC is divided into capillary endothelial cell or smooth muscle cell; Be connected and synthesize macromolecule branchSupport, proceeds by said structure the in vitro culture of pulsing, and alternately changes containing the DMEM of VEGF, containing the DMEM of HGF, containing nephrocyteThe DMEM of derivant and general DMEM, realize organized stable, the development of vascularization of nephrocyte and the connection between the twoSystem.
5) according to the step 4 of embodiment 9) carry out induction generation and the ephritis drug screening of ephritis.
Embodiment 12: prepare a kind of personalization bionical vascularization renal carcinoma tissue and be applied to the drug screening of kidney
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC), buy kidney cancer cell (RCC), trainingAfter foster going down to posterity, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, obtain mass body volume concentrations and be 10%The fibrinogen solution that gelatin solution and mass body volume concentrations are 1%, with gelatin solution and fibrinogen solution volume ratio 1:After 1 mixing, obtain natural polymer stand-by; ADSC and RCC are centrifugal respectively the most at last, and sneak into respectively in natural polymer solution,Obtaining cell concentration is 3 × 106The natural polymer solution containing ADSC of individual/mL, and cell concentration is 3 × 106Individual/mL'sContaining the natural polymer solution of RCC.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) prepare natural polymer sodium alginate branch bloodPipe holder, crosslinked with CaCl2.
3) step 3 of the preparation of macromolecule shell and polymeric isolation layer: embodiment 1) prepare natural polymer alginic acidSodium shell and separation layer, crosslinked with CaCl2.
4) the final establishment of external bionic model: utilize compound many shower nozzles 3D printing technique by the natural polymer containing RCCBranch vessel path or computer model along design in advance carry out 3 D-printing, and interval reserves 50% branch vessel space notGive cell assembling; Utilize afterwards compound many shower nozzles 3D printing technique by containing the natural polymer of ADSC along above-mentioned branch vessel inner sideReserved path print, and inside printable layer reserving hole channel; Utilize pulsation system supply MEM nutrient solution to stablize kidney cancer cell and make itSystematism; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and HGF (HGF), induction ADSC dividesTurn to capillary endothelial cell or smooth muscle cell; Said structure is proceeded to the in vitro culture of pulsing, alternately change and containThe MEM of VEGF, containing MEM and the general MEM of HGF, realize the development of organized stable, vascularization of kidney cancer cell and the twoBetween contact.
5) kidney drug screening: the biological indicator of detection record formed body is (as urea nitrogen, creatinine, alkaline phosphatase, breastAcidohydrogenase and vascularization expression); Utilize pulsation system supply with curative drug as Sorafenib, shellfish cut down strain monoclonal antibody orMedicine newly developed, records and analyzes biological indicator, carries out drug screening.
Embodiment 13 prepares a kind of personalization bionical vascularization lymph cancer tissue and is applied to the drug sieve of lymph cancerChoosing
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC), buy lymphocytic cancer cell (EL4),After subculture, prepare cell suspending liquid; Natural polymer powder is mixed with PBS solution, and obtaining mass body volume concentrations is 1%Fibrinogen solution, ADSC and EL4 are centrifugal respectively the most at last, and sneak into respectively in natural polymer solution, obtain cellConcentration is 2 × 105The natural polymer solution containing ADSC of individual/mL, and cell concentration is 2 × 105The sky containing EL4 of individual/mLSo Polymer Solution.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) prepare synthesis of natural macromolecular fibre proteinogenBranch vessel support, with the fibrin ferment polymerization of 100U/mL.
3) step 3 of the preparation of macromolecule shell and polymeric isolation layer: embodiment 1) prepare natural polymer subbundle eggWhite former shell and separation layer, with the fibrin ferment polymerization of 100U/mL.
4) utilize compound many shower nozzles 3D printing technique by the branch vessel path of design in advance, natural polymer edge containing EL4Or computer model carries out 3 D-printing, interval reserves 50% branch vessel space and refuses cell assembling; Utilize afterwards compound manyShower nozzle 3D printing technique will be printed along reserved path inside above-mentioned branch vessel containing the natural polymer of ADSC, and in printable layerSide reserving hole channel; Utilize pulsation system supply training MEM nutrient solution to stablize lymphocytic cancer cell and make its systematism; Utilize pulsation system to supply withThe angiogenesis factor (VEGF) and HGF (HGF), induction ADSC be divided into capillary endothelial cell orSmooth muscle cell; Said structure is proceeded to the in vitro culture of pulsing, alternately change containing the DMEM of VEGF, containing the DMEM of HGF andGeneral DMEM, realizes organized stable, the development of vascularization of lymphocytic cancer cell and the contact between the two.
5) lymph cancer drug screening: the biological indicator of detection record formed body is (as lactate dehydrogenase, alkaline phosphataseEnzyme, tumor giant cell and vascularization expression); Utilize pulsation system to supply with curative drug as adriamycin, bleomycin, ChangchunFlower alkali, first nitrogen miaow ammonia or medicine newly developed, record and analyze biological indicator, carries out drug screening.
Embodiment 14: the little ear tissue of a kind of personalized bionical vascularization of preparation
1) preparation of celliferous hydrogel: extract adipose derived stem cells of rats (ADSC) and cartilage cell (CHO), preparation is thinBorn of the same parents' suspension; Natural polymer powder is mixed with DMEM nutrient solution, obtain mass body volume concentrations and be 5% gelatin solution, finalBy centrifugal respectively to ADSC and CHO suspension, sneak into respectively in natural polymer solution, obtain 3 × 106Individual/mL contains ADSC'sNatural polymer solution, and 3 × 106The natural polymer solution containing CHO of individual/mL.
2) preparation of branch vessel support: according to the step 2 of embodiment 1) the synthetic macromolecule ABS branch vessel of preparation props upFrame.
3) preparation of macromolecule shell and polymeric isolation layer: material is that mass body volume concentrations is Isosorbide-5-Nitrae-dioxy of 3% PUSix ring solution, in solution, sneak into heparin and leptin, utilize multiinjector spraying nozzle assembly, by this solution spraying to hydrogel layer itBetween; Utilize spray nozzle spraying PBS solution, extraction, removal TEG, obtain porous PU separation layer; Obtain hydrogel-PU layerAlternating structure, be protective layer; This solution spraying, to structure periphery, obtains PU shell the most at last.
4) utilize compound many shower nozzles 3D printing technique by the branch vessel path of design in advance, natural polymer edge containing CHOOr computer model carries out 3 D-printing, interval reserves 50% branch vessel space and refuses cell assembling; Utilize afterwards compound manyShower nozzle 3D printing technique will be printed along reserved path inside above-mentioned branch vessel containing the natural polymer of ADSC, and in printable layerSide reserving hole channel; Formed body is immersed to PBS solution, and TEG organic solvent is removed in extraction; Utilizing pulsation system to supply with cultivatesLiquid is stablized cartilage cell CHO makes its systematism; Utilize pulsation system to supply with the angiogenesis factor (VEGF) and liver cell lifeThe long factor (HGF), induction ADSC is divided into capillary endothelial cell or smooth muscle cell; Said structure is proceeded to pulsationIn vitro culture, alternately changes containing the DMEM of VEGF, containing DMEM and the general DMEM of HGF, realizes the organized steady of Chinese hamster ovary celIThe development of vascularization and the contact between the two calmly.
Claims (9)
1. the bionical composite construction of personalization, is characterized in that: the bionical composite construction of described personalization comprises macromolecule shell(106), branch vessel support (102), district of functional organization (103), district of vascular tissue (104) and polymeric isolation layer (401); InstituteThe district of functional organization (103) stating is for containing histiocytic hydrogel layer; Described district of vascular tissue (104) is for careful containing blood vessel kindBorn of the same parents' hydrogel layer; Arrange at space-alternating district of described functional organization (103) and district of vascular tissue (104); Described macromoleculeSeparation layer (401) is intervally arranged in district of functional organization (103) and district of vascular tissue (104), and by district of functional organization (103) andDistrict of vascular tissue (104) is divided into multiple zonules; Described branch vessel support (102) comprises two of arteries and vein blood vesselsPoint, arteries is containing at least one entrance and several branches, and vein blood vessel is containing at least one outlet and several branches; DescribedBranch vessel support (102) adopt synthesized polymer material, branch vessel support (102) through macromolecule shell (106),Between district of functional organization (103), district of vascular tissue (104) and polymeric isolation layer (401); Described macromolecule shell (106)Adopt natural polymer or synthesized polymer material; District of described functional organization containing histiocytic hydrogel layer and vascular tissueThe hydrogel layer containing blood vessel seed cell in district is natural polymer hydrogel, is compounded with thin in described natural polymer hydrogelThe intracellular growth factor;
The internal diameter of described branch vessel support is 10 μ m～10mm, and wall thickness is 100 μ m～2mm; Described polymeric isolation layer and heightDividing the bed thickness of subshell is 0.1 μ m～2mm.
2. the personalized bionical composite construction of one as claimed in claim 1, is characterized in that: this natural polymer hydrogel isGelatin, sodium alginate, fibrinogen, collagen, matrigel, carragheen, shitosan, agar, hyaluronic acid, elastin laminin and layerAt least one in sticky element; In described natural polymer hydrogel, be also compounded with in heparin, leptin, taxol and Radix Astragali anti-cancer powderAt least one.
3. the personalized bionical composite construction of one as claimed in claim 2, is characterized in that: described natural polymer hydrogelMass body volume concentrations be 0.1～40%; Described blood vessel seed cell and histiocytic concentration are 1 × 103～1×108Individual/ML; The histocyte in district of described functional organization is the one in adult tissue's cell, adult stem cell and cancer cell; Described blood vesselThe blood vessel seed cell of organizing district is vascular endothelial cell, fat stem cell, mesenchymal stem cells MSCs, navel blood stem cell, boneAt least one in marrow stem cell, embryonic stem cell and induced multi-potent stem cells.
4. a preparation method for personalized bionical composite construction as claimed in claim 1, it is characterized in that the method comprise withLower step:
1) design branch vessel support threedimensional model, divides two groups and imitates respectively arteries and vein blood vessel;
2) by synthesized polymer material, prepare containing histiocytic hydrogel, prepare containing the water of blood vessel seed cellGel and the synthetic Polymer Solution preparing are loaded in the different shower nozzles of compound many shower nozzles 3 D-printing equipment;
3) prepare branch vessel support: according to step 1) threedimensional model, utilize compound many shower nozzles 3 D-printing equipment to extrude and closeBecome Polymer Solution, and freeze-drying removal organic solvent, or melt extrude synthesized polymer material, obtain branch vessel support;
4) prepare district of functional organization: utilize compound many shower nozzles 3 D-printing equipment successively to print to prepare containing histiocytic waterGel, printing path is along branch vessel support, and print at interval, obtains preliminary three-dimensional structure;
5) prepare district of vascular tissue: utilize compound many shower nozzles 3 D-printing equipment successively to print, by prepare containing blood vessel seedThe hydrogel of cell is filled in the space of preliminary three-dimensional structure, three-dimensional structure in the middle of obtaining;
6) prepare polymeric isolation layer and macromolecule shell: in step 4) and step 5) successively in print procedure, printing one deckOr after some layers, utilize the synthetic Polymer Solution of compound many shower nozzles 3 D-printing equipment spraying and extract organic solvent, or melting is squeezedGo out synthesized polymer material, form polymeric isolation layer, obtain the alternately knot of celliferous hydrogel layer and polymeric isolation layerStructure body; Synthesize Polymer Solution and extract organic solvent in the peripheral spraying of alternating structure body, or melt extruding synthetic macromolecule materialMaterial, forms macromolecule shell, obtains personalized bionical composite construction precursor;
7) provide growth factor to the bionical composite construction precursor of above-mentioned personalization, realize the vascularization in district of vascular tissue, make functionOrganize district's formative tissue, and make the cell in district of vascular tissue and district of functional organization produce contact, finally obtain described personalizationBionical composite construction.
5. the preparation method of the bionical composite construction of a kind of personalization as claimed in claim 4, is characterized in that: described compound manyThe employing of shower nozzle 3 D-printing equipment melt extrudes forming technique or low temperature depositing forming technique is prepared branch vessel support, high scoreSub-separation layer and macromolecule shell; When melt extruding forming technique, employing prepares branch vessel support, polymeric isolation layer and heightWhile dividing subshell, described synthesized polymer material is acrylonitrile-butadiene-styrene (ABS), polyurethane, polytetrafluoroethylene (PTFE) and carbohydrateIn at least one; Prepare branch vessel support, polymeric isolation layer and macromolecule shell when adopting low temperature depositing forming techniqueTime, the solute of described synthetic Polymer Solution is polyurethane, Merlon, polyureas ester, PGA, poly-succinic ester, poly-At least one in ether-ether, polyethylene glycol, PLA, polycaprolactone, Poly(D,L-lactide-co-glycolide and polyhydroxy acid ester,The solvent of described synthetic Polymer Solution is TEG or Isosorbide-5-Nitrae-dioxane.
6. the preparation method of the bionical composite construction of a kind of personalization as claimed in claim 4, is characterized in that: described synthetic heightThe mass body volume concentrations of molecular solution is 1%～30%; In described synthetic Polymer Solution, be compounded with anticoagulin, this anti-freezingBlood factor is heparin or taxol.
7. personalized bionical composite construction, for a method for drug screening, is characterized in that the party as claimed in claim 1Method comprises the steps:
1) if the histocyte in district of functional organization adopts adult tissue's cell or adult stem cell, to the bionical composite junction of personalizationStructure is supplied with the nutrient solution containing pathogenic medicine, makes the bionical composite construction of above-mentioned personalization produce pathological symptom; If district of functional organizationHistocyte adopt cancer cell, do not do pathogenic processing;
2) the bionical composite construction of personalization is supplied with to the nutrient solution containing variety classes and various dose curative drug, in real time or afterThe continuous nutrient solution of collecting;
3) analyze the above-mentioned nutrient solution of having collected, the kind of obtaining medical treatment property medicine, the work of dosage to described personalized composite constructionWith, by detecting, contrasting biological indicator, judge the effect of medicine.
8. the bionical composite construction of a kind of personalization as claimed in claim 7, for the method for drug screening, is characterized in that: instituteStating biological indicator is in carbohydrate lipid metabolism index, liver function index, renal function index, cancer cell index and vascularization indexAt least one.
9. the bionical composite construction of a kind of personalization as claimed in claim 7, for the method for drug screening, is characterized in that: instituteStating pathological symptom is diabetes, organ inflammation, organ failure or tumour.
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| CN104232484A (en) * | 2014-09-11 | 2014-12-24 | 浙江大学 | Cell co-culture model and preparation method |
| CN104490489B (en) * | 2014-12-02 | 2017-02-01 | 淮安皓运生物科技有限公司 | Method for preparing tissue engineering blood vessel based on 3D bioprinting technology |
| TWI741980B (en) * | 2015-04-07 | 2021-10-11 | 大陸商四川藍光英諾生物科技股份有限公司 | Biological brick and its use |
| DK3280463T3 (en) | 2015-04-07 | 2021-08-02 | Revotek Co Ltd | Compositions for cell-based three-dimensional printing |
| WO2016161941A1 (en) * | 2015-04-07 | 2016-10-13 | Sichuan Revotek Co., Ltd. | Bio-blocks comprising endothelial cells and methods of use thereof |
| CN108079384B (en) * | 2015-04-07 | 2021-07-06 | 四川蓝光英诺生物科技股份有限公司 | Biological brick containing endothelial cells and application thereof |
| CN104931683B (en) * | 2015-05-21 | 2017-01-04 | 清华大学 | A kind of cardiac muscular tissue sensor and the preparation method of cardiac muscular tissue's chip |
| CN106606804B (en) * | 2015-10-22 | 2020-05-12 | 四川蓝光英诺生物科技股份有限公司 | Method for preparing composite structure |
| CN105688281A (en) * | 2016-03-08 | 2016-06-22 | 北京航空航天大学 | Vascularized artificial tissue organ construction method combining cell three-dimensional printing and dielectrophoresis adsorption |
| CN105688280A (en) * | 2016-03-08 | 2016-06-22 | 北京航空航天大学 | Three-dimensional cell assembly method based on dielectrophoresis adsorption principle |
| JP6755952B2 (en) * | 2016-09-14 | 2020-09-16 | レボテック カンパニー，リミティド | Artificial tissue precursors and methods for preparing them |
| US11439731B2 (en) | 2016-09-14 | 2022-09-13 | Revotek Co., Ltd. | Artificial tissue progenitor and method for preparing the same |
| CN106434562B (en) * | 2016-09-19 | 2020-03-13 | 广州迈普再生医学科技股份有限公司 | Brain tumor in-vitro model for three-dimensional biological printing and construction method thereof |
| WO2019246416A1 (en) * | 2018-06-21 | 2019-12-26 | Yale University | Bioartificial vascular pancreas |
| CN110118863A (en) * | 2018-09-21 | 2019-08-13 | 苏州永沁泉智能设备有限公司 | A kind of high-throughput drug automatic splinter screening device for screening and method |
| CN111197024B (en) * | 2018-11-16 | 2023-08-18 | 杭州捷诺飞生物科技股份有限公司 | Pancreatic-like structure, construction method and application thereof |
| CN115970056A (en) * | 2023-01-31 | 2023-04-18 | 陕西师范大学 | 3D printing material for bone repair, bone repair material, and preparation method and application thereof |
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| CN101623515A (en) * | 2009-07-31 | 2010-01-13 | 清华大学 | Method for preparing complicated tissue organ precursor with multilayer structure |
| CN101775431B (en) * | 2009-12-25 | 2013-09-25 | 杭州电子科技大学 | Method for high content screening of therapeutic drugs for diabetes |
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